The Interactive Fly

Zygotically transcribed genes

Oncogenes and Tumor Suppressors

  • Regulation of cell growth by Notch signaling and its differential requirement in normal vs. tumor-forming stem cells in Drosophila
  • Transformed Drosophila cells evade diet-mediated insulin resistance through wingless signaling
  • Bunched and Madm function downstream of Tuberous Sclerosis Complex to regulate the growth of intestinal stem cells in Drosophila
  • The asymmetrically segregating lncRNA cherub is required for transforming stem cells into malignant cells
  • Drosophila tumor suppressor gene prevents tonic TNF signaling through receptor N-glycosylation
  • Warburg effect metabolism drives neoplasia in a Drosophila genetic model of epithelial cancer
  • The ligand Sas and its receptor PTP10D drive tumour-suppressive cell competition
  • Structural basis for the activation of the deubiquitinase Calypso by the Polycomb protein ASX
  • Evidence for a novel function of Awd in maintenance of genomic stability
  • The transcription factor Ets21C drives tumor growth by cooperating with AP-1
  • Cytoneme-mediated signaling essential for tumorigenesis
  • A positive feedback loop between Myc and aerobic glycolysis sustains tumor growth in a Drosophila tumor model
  • Spz/Toll-6 signal guides organotropic metastasis in Drosophila
  • Coopted temporal patterning governs cellular hierarchy, heterogeneity and metabolism in Drosophila neuroblast tumors
  • The transcription factor spalt and human homologue SALL4 induce cell invasion via the dMyc-JNK pathway in Drosophila
  • Notch mediates inter-tissue communication to promote tumorigenesis
  • Genome Wide Screen for Context-Dependent Tumor Suppressors Identified Using in Vivo Models for Neoplasia in Drosophila
  • A Genetic Analysis of Tumor Progression in Drosophila Identifies the Cohesin Complex as a Suppressor of Individual and Collective Cell Invasion
  • NatB regulates Rb mutant cell death and tumor growth by modulating EGFR/MAPK signaling through the N-end rule pathways
  • A Forward Genetic Approach to Mapping a P-Element Second Site Mutation Identifies DCP2 as a Novel Tumour Suppressor in Drosophila melanogaster
  • Context-Dependent Tumorigenic Effect of Testis-Specific Mitochondrial Protein Tiny Tim 2 in Drosophila Somatic Epithelia
  • A Drosophila model of oral peptide therapeutics for adult Intestinal Stem Cell tumors
  • Role for phagocytosis in the prevention of neoplastic transformation in Drosophila
  • Systemic muscle wasting and coordinated tumour response drive tumourigenesis
  • Transcriptional repression of Myc underlies the tumour suppressor function of AGO1 in Drosophila
  • Hyperinsulinemia drives epithelial tumorigenesis by abrogating cell competition
  • Mammalian Snail-induced Claudin-11 prompts collective migration for tumour progression
  • Cross-species identification of PIP5K1-, splicing- and ubiquitin-related pathways as potential targets for RB1-deficient cells
  • Aneuploidy facilitates dysplastic and tumorigenic phenotypes in the Drosophila gut
  • Methionine restriction breaks obligatory coupling of cell proliferation and death by an oncogene Src in Drosophila
  • The H3.3K27M oncohistone antagonizes reprogramming in Drosophila

    Ras oncogene
  • Mutations in the Drosophila tricellular junction protein M6 synergize with Ras(V12) to induce apical cell delamination and invasion
  • Dissemination of Ras(V12)-transformed cells requires the mechanosensitive channel Piezo
  • The level of oncogenic Ras determines the malignant transformation of Lkb1 mutant tissue in vivo
  • Cooperation between oncogenic Ras and wild-type p53 stimulates STAT non-cell autonomously to promote tumor radioresistance
  • Misshapen Disruption Cooperates with Ras(V12) to Drive Tumorigenesis

  • Oncogenes and Tumor Supressors

    Regulation of cell growth by Notch signaling and its differential requirement in normal vs. tumor-forming stem cells in Drosophila

    Cancer stem cells (CSCs) are postulated to be a small subset of tumor cells with tumor-initiating ability that shares features with normal tissue-specific stem cells. The origin of CSCs and the mechanisms underlying their genesis are poorly understood, and it is uncertain whether it is possible to obliterate CSCs without inadvertently damaging normal stem cells. This study shows that a functional reduction of eukaryotic translation initiation factor 4E (eIF4E) in Drosophila specifically eliminates CSC-like cells in the brain and ovary without having discernible effects on normal stem cells. Brain CSC-like cells can arise from dedifferentiation of transit-amplifying progenitors upon Notch hyperactivation. eIF4E is up-regulated in these dedifferentiating progenitors, where it forms a feedback regulatory loop with the growth regulator dMyc to promote cell growth, particularly nucleolar growth, and subsequent ectopic neural stem cell (NSC) formation. Cell growth regulation is also a critical component of the mechanism by which Notch signaling regulates the self-renewal of normal NSCs. These findings highlight the importance of Notch-regulated cell growth in stem cell maintenance and reveal a stronger dependence on eIF4E function and cell growth by CSCs, which might be exploited therapeutically (Song, 2011).

    The CSC hypothesis was initially developed based on studies in mammalian systems. Various studies have supported the notion that CSCs share many functional features with normal stem cells, such as signaling molecules, pathways, and mechanisms governing their self-renewal versus differentiation choice. However, the cellular origin of CSCs and the molecular and cellular mechanisms underlying their development or genesis remain poorly understood. It has been proposed that CSCs could arise from (1) an expansion of normal stem cell niches, (2) normal stem cells adapting to different niches, (3) normal stem cells becoming niche-independent, or (4) differentiated progenitor cells gaining stem cell properties. This study has shown that in the Drosophila larval brain, CSCs can arise from the dedifferentiation of transit-amplifying progenitor cells back to a stem cell-like state. Importantly, eIF4E was identified as a critical factor involved in this dedifferentiation process. More significantly, it was shown that reduction of eIF4E function can effectively prevent the formation of CSCs without affecting the development or maintenance of normal stem cells. This particular dependence on eIF4E function by CSCs appears to be a general theme, as reduction of eIF4E function also effectively prevented the formation of CSCs, but not normal GSCs, in the fly ovary. These findings may have important implications for stem cell biology and cancer biology, in terms of both mechanistic understanding and therapeutic intervention (Song, 2011).

    This study also offers mechanistic insights into the cellular processes leading to the dedifferentiation of progenitors back to stem cells. In Drosophila type II NB clones with overactivated N signaling, ribosome biogenesis within ectopic NBs appears to be faster than in normal NBs, as shown by the fact that the ratio of nucleolar to cellular volume of the ectopic NBs is approximately fivefold higher than that of normal NBs. The faster growth rate is accompanied by the up-regulation of dMyc and eIF4E and appears to be essential for transit-amplifying progenitors to undergo complete dedifferentiation back to a stem cell-like state. When the function of cell growth-promoting factors such as eIF4E is attenuated, the faster cell growth of ectopic NBs can no longer be sustained and the dedifferentiation process stalls. As a result, brain tumor formation caused by uncontrolled production of ectopic NBs is suppressed. In contrast, normal NBs, which presumably have relatively lower requirements for cell growth and hence eIF4E function, maintain their stem cell fate and development under similar conditions. Therefore, a potential key to a successful elimination of CSC-induced tumors would be to find the right level of functional reduction in eIF4E, which causes minimal effects on normal stem cells but effectively obliterates CSCs. An ongoing clinical trial with Ribavirin in treating acute myeloid leukemia (AML), a well-characterized CSC-based cancer, demonstrated exciting proof of principle that such a strategy is feasible. The current version of Ribavirin, however, has certain limitations, such as its poor specificity and the high dosage (micromolar range) required for effective treatment. Thus, more specific and effective eIF4E inhibitors are urgently needed. Drug treatment experiments with Ribavirin validated Drosophila NBs as an excellent CSC model for searching further improved drugs. More importantly, the nuclear interaction between eIF4E and Myc unraveled by the biochemical analysis not only provides a new mechanistic explanation for the synergistic effects of eIF4E and Myc in tumorigenesis, but also sheds new light on how to rationally optimize drug design and therapy for treating CSC-based cancer (Song, 2011).

    The results offer new information on how N signaling helps specify and maintain NSC fate. N signaling regulates stem cell behavior in various tissues of diverse species. However, it remains unclear how differential N signaling determines distinct cell fate within the stem cell hierarchy. This study demonstrates that N signaling maintains Drosophila NSC fate at least in part through promoting cell growth. The following evidence supports that cell growth, but not cell fate, change is the early and primary effect of N signaling inhibition in type II NBs: (1) Pros expression is not immediately turned on in spdo mutant NBs with reduced cell sizes. Instead, it gradually increases during the course of spdo mutant NB divisions. (2) Up-regulation of Pros is not the cause of stem cell fate loss in spdo mutant NBs, as shown by spdo pros double-mutant analysis. (3) Cell growth defects precede the up-regulation of Ase expression in aph-1 (coding for a component of γ secretase) mutant NBs. (4) Promotion of cell growth, and particularly nucleolar growth, by dMyc is sufficient to prevent NB loss caused by N inhibition. At the molecular level, N signaling appears to regulate the transcription of dMyc, which in turn up-regulates the transcription of eIF4E. Such a transcriptional cascade and feedback regulation of dMyc activity by eIF4E may help to sustain and amplify the activity of the Notch-dMyc-eIF4E molecular circuitry. Hence, differential N signaling within the lineage can lead to different cell growth rates, which partially determine differential cell fates. Consistent with this notion, knockdown of both eIF4E and dMyc results in defects of NB cell growth and loss of stem cell fate (Song, 2011).

    While many signaling pathways and molecules have been implicated in the maintenance of stem cell identity, the question of how a stem cell loses its 'stemness' at the cellular level remains poorly understood. A stem cell may lose its stem cell fate by undergoing a symmetric division to yield two daughter cells that are both committed to differentiation or through cell death. Earlier studies provided intriguing hints that cell growth and translational regulation could influence stem cell maintenance in the Drosophila ovary. Detailed clonal analyses of NSCs over multiple time points provides direct evidence that a NSC with impaired N signaling will gradually lose its identity due to a gradual slowing down of cell growth and loss of cell mass. Remarkably, such loss of stem cell fate can be prevented when cell growth is restored by dMyc, but not Rheb, overexpression, demonstrating the functional significance of regulated cell growth, particularly nucleolar growth, in stem cell maintenance. More importantly, this information offers clues on how to specifically eliminate tumor-initiating stem cells. The current studies suggest that a stem cell, normal or malignant, has to reach a certain growth rate in order to acquire and maintain its stemness, presumably because when the stem cell grows below such a threshold, its proliferative capacity becomes too low, whereas the concentration of differentiation-promoting factors becomes too high to be compatible with the maintenance of stem cell fate. Consistent with this notion are the strong correlation between the expression of ribosomal proteins and cellular proliferation as well as the correlation between the reduction of NB sizes and the up-regulation of differentiation-promoting factor Pros or Ase in different developmental contexts (Song, 2011).

    The results also provide new insights into how the evolutionarily conserved tripartite motif and Ncl-1, HT2A, and Lin-41 (TRIM-NHL) domain proteins regulate stem cell homeostasis. The TRIM-NHL protein family, to which Brat and Mei-P26 belong, include evolutionarily conserved stem cell regulators that prevent ectopic stem cell self-renewal by inhibiting Myc. However, the downstream effectors of the TRIM-NHL proteins remain largely unknown. This study has identified eIF4E as such a factor. NB-specific knockdown of eIF4E completely suppresses the drastic brain tumor phenotype caused by loss of Brat. Interestingly, eIF4E knockdown is even more effective than dMyc knockdown in this regard. N signaling and Brat have been proposed to act in parallel in regulating Drosophila type II NB homeostasis. However, at the molecular level, how deregulation of these two rather distinct pathways causes similar brain tumor phenotypes remain largely unknown. The results suggest that these two pathways eventually converge on the dMyc-eIF4E regulatory loop to promote cell growth and stem cell fate. N overactivation and loss of Brat both result in up-regulation of eIF4E and dMyc in transit-amplifying progenitors, accelerating their growth rates and helping them acquire stem cell fate. Consistent with a general role of eIF4E and dMyc in stem cell regulation, it was shown that partial reduction of eIF4E or dMyc function in the Drosophila ovary effectively rescues the ovarian tumor phenotype due to the loss of Mei-P26. The vertebrate member of the TRIM-NHL family, TRIM32, is shown to suppress the stem cell fate of mouse neural progenitor cells, partially through degrading Myc. Whether eIF4E acts as a downstream effector of TRIM32 in balancing stem cell self-renewal versus differentiation in mammalian tissues awaits future investigation (Song, 2011).

    Transformed Drosophila cells evade diet-mediated insulin resistance through wingless signaling

    Cancer cells demand excessive nutrients to support their proliferation but how cancer cells sense and promote growth in the nutrient favorable conditions remain incompletely understood. Epidemiological studies have indicated that obesity is a risk factor for various types of cancers. Feeding Drosophila a high dietary sugar was previously demonstrated to not only direct metabolic defects including obesity and organismal insulin resistance, but also transform Ras/Src-activated cells into aggressive tumors. This study demonstrates that Ras/Src-activated cells are sensitive to perturbations in the Hippo signaling pathway. Evidence that nutritional cues activate Salt-inducible kinase, leading to Hippo pathway downregulation in Ras/Src-activated cells. The result is Yorkie-dependent increase in Wingless signaling, a key mediator that promotes diet-enhanced Ras/Src-tumorigenesis in an otherwise insulin-resistant environment. Through this mechanism, Ras/Src-activated cells are positioned to efficiently respond to nutritional signals and ensure tumor growth upon nutrient rich condition including obesity (Hirabayashi, 2015).

    The prevalence of obesity is increasing globally. Obesity impacts whole-body homeostasis and is a risk factor for severe health complications including type 2 diabetes and cardiovascular disease. Accumulating epidemiological evidence indicates that obesity also leads to elevated risk of developing several types of cancers. However, the mechanisms that link obesity and cancer remain incompletely understood. Using Drosophila, a whole-animal model system has been developed to study the link between diet-induced obesity and cancer: this model has provided a potential explanation for how obese and insulin resistant animals are at increased risk for tumor progression (Hirabayashi, 2015).

    Drosophila fed a diet containing high levels of sucrose (high dietary sucrose or ‘HDS') developed sugar-dependent metabolic defects including accumulation of fat (obesity), organismal insulin resistance, hyperglycemia, hyperinsulinemia, heart defects and liver (fat body) dysfunctions. Inducing activation of oncogenic Ras and Src together in the Drosophila eye epithelia led to development of small benign tumors within the eye epithelia. Feeding animals HDS transformed Ras/Src-activated cells from benign tumor growths to aggressive tumor overgrowth with tumors spread into other regions of the body (Hirabayashi, 2013). While most tissues of animals fed HDS displayed insulin resistance, Ras/Src-activated tumors retained insulin pathway sensitivity and exhibited an increased ability to import glucose. This is reflected by increased expression of the Insulin Receptor (InR), which was activated through an increase in canonical Wingless (Wg)/dWnt signaling that resulted in evasion of diet-mediated insulin resistance in Ras/Src-activated cells. Conversely, expressing a constitutively active isoform of the Insulin Receptor in Ras/Src-activated cells (InR/Ras/Src) was sufficient to elevate Wg signaling, promoting tumor overgrowth in animals fed a control diet. These results revealed a circuit with a feed-forward mechanism that directs elevated Wg signaling and InR expression specifically in Ras/Src-activated cells. Through this circuit, mitogenic effects of insulin are not only preserved but are enhanced in Ras/Src-activated cells in the presence of organismal insulin resistance (Hirabayashi, 2015).

    These studies provide an outline for a new mechanism by which tumors evade insulin resistance, but several questions remain: (1) how Ras/Src-activated cells sense the organism's increased insulin levels, (2) how nutrient availability is converted into growth signals, and (3) the trigger for increased Wg protein levels, a key mediator that promotes evasion of insulin resistance and enhanced Ras/Src-tumorigenesis consequent to HDS. This study identifies the Hippo pathway effector Yorkie (Yki) as a primary source of increased Wg expression in diet-enhanced Ras/Src-tumors. Ras/Src-activated cells are sensitized to Hippo signaling, and even a mild perturbation in upstream Hippo pathway is sufficient to dominantly promote Ras/Src-tumor growth. Functional evidence is provided that increased insulin signaling promotes Salt-inducible kinases (SIKs) activity in Ras/Src-activated cells, revealing a SIKs-Yki-Wg axis as a key mediator of diet-enhanced Ras/Src-tumorigenesis. Through this pathway, Hippo-sensitized Ras/Src-activated cells are positioned to efficiently respond to insulin signals and promote tumor overgrowth. These mechanisms act as a feed-forward cassette that promotes tumor progression in dietary rich conditions, evading an otherwise insulin resistant state (Hirabayashi, 2015).

    Previously work has demonstrated that Ras/Src-activated cells preserve mitogenic effects of insulin under the systemic insulin resistance induced by HDS-feeding of Drosophila (Hirabayashi, 2013). Evasion of insulin resistance in Ras/Src-activated cells is a consequence of a Wg-dependent increase in InR gene expression (Hirabayashi, 2013). This study identified the Hippo pathway effector Yki as a primary source of the Wnt ortholog Wg in diet-enhanced Ras/Src-tumors. Mechanistically, functional evidence is provided that activation of SIKs promotes Yki-dependent Wg-activation and reveal a SIK-Yki-Wg-InR axis as a key feed-forward signaling pathway that underlies evasion of insulin resistance and promotion of tumor growth in diet-enhanced Ras/Src-tumors (Hirabayashi, 2015).

    In animals fed a control diet, at most a mild increase was observed in Yki reporter activity within ras1G12V;csk-/- cells. A previous report indicates that activation of oncogenic Ras (ras1G12V) led to slight activation of Yki in eye tissue. Activation of Src through over-expression of the Drosophila Src ortholog Src64B has been shown to induce autonomous and non-autonomous activation of Yki. In contrast, inducing activation of Src through loss of csk (csk-/-) failed to elevate diap1 expression. The results indicate that activation of Yki is an emergent property of activating Ras plus Src (ras1G12V;csk-/-). However, this level of Yki-activation was not sufficient to promote stable tumor growth of Ras/Src-activated cells in the context of a control diet: Ras/Src-activated cells were progressively eliminated from the eye tissue (Hirabayashi, 2013). It was, however, sufficient to sensitize Ras/Src-activated cells to upstream Hippo pathway signals: loss of a genetic copy of ex-which was not sufficient to promote growth by itself-dominantly promoted tumor growth of Ras/Src-activated cells even in animals fed a control diet. These data provide compelling evidence that Ras/Src-transformed cells are sensitive to upstream Hippo signals (Hirabayashi, 2015).

    SIK was recently demonstrated to phosphorylate Sav at Serine-413, resulting in dissociation of the Hippo complex and activation of Yki (Wehr, 2013). SIKs are required for diet-enhanced Ras/Src-tumor growth in HDS. Conversely, expression of a constitutively activated isoform of SIK was sufficient to promote Ras/Src-tumor overgrowth even in a control diet. Mammalian SIKs are regulated by glucose and by insulin signaling. However, a recent report indicated that glucagon but not insulin regulates SIK2 activity in the liver. The current data demonstrate that increased insulin signaling is sufficient to promote SIK activity through Akt in Ras/Src-activated cells. It is concluded that SIKs couple nutrient (insulin) availability to Yki-mediated evasion of insulin resistance and tumor growth, ensuring Ras/Src-tumor growth under nutrient favorable conditions (Hirabayashi, 2015).

    The results place SIKs as key sensors of nutrient and energy availability in Ras/Src-tumors through increased insulin signaling and, hence, increased glucose availability. SIK activity promotes Ras/Src-activated cells to efficiently respond to upstream Hippo signals, ensuring tumor overgrowth in organisms that are otherwise insulin resistant. One interesting question is whether this mechanism is relevant beyond the context of an obesity-cancer connection: both Ras and Src have pleiotropic effects on developmental processes including survival, proliferation, morphogenesis, differentiation, and invasion, and these mechanisms may facilitate these processes under nutrient favorable conditions. From a treatment perspective the current data highlight SIKs as potential therapeutic targets. Limiting SIK activity through compounds such as HG-9-91-01 may break the connection between oncogenes and diet, targeting key aspects of tumor progression that are enhanced in obese individuals (Hirabayashi, 2015).

    Bunched and Madm function downstream of Tuberous Sclerosis Complex to regulate the growth of intestinal stem cells in Drosophila

    The Drosophila adult midgut contains intestinal stem cells that support homeostasis and repair. This study shows that the leucine zipper protein Bunched and the adaptor protein MLF1-adaptor molecule (Madm) are novel regulators of intestinal stem cells. MARCM mutant clonal analysis and cell type specific RNAi revealed that Bunched and Madm were required within intestinal stem cells for proliferation. Transgenic expression of a tagged Bunched showed a cytoplasmic localization in midgut precursors, and the addition of a nuclear localization signal to Bunched reduced its function to cooperate with Madm to increase intestinal stem cell proliferation. Furthermore, the elevated cell growth and 4EBP phosphorylation phenotypes induced by loss of Tuberous Sclerosis Complex or overexpression of Rheb were suppressed by the loss of Bunched or Madm. Therefore, while the mammalian homolog of Bunched, TSC-22, is able to regulate transcription and suppress cancer cell proliferation, these data suggest the model that Bunched and Madm functionally interact with the TOR pathway in the cytoplasm to regulate the growth and subsequent division of intestinal stem cells (Nie, 2015).

    Homeostasis and regeneration of an adult tissue is normally supported by resident stem cells. Elucidation of the mechanisms that regulate stem cell-mediated homeostasis is important for the development of therapeutics for various diseases. The intestine with fast cell turnover rate supported by actively proliferating stem cells is a robust system to study tissue homeostasis. In the mouse intestine, two interconverting intestinal stem cell (ISC) populations marked by Bmi1 and Lgr5 located near the crypt base can replenish cells of various lineages along the crypt-villus axis Furthermore, recent data suggest that Lgr5+ cells are the main stem cell population and that immediate progeny destined for the secretory lineage can revert to Lgr5+ stem cells under certain conditions [6, 7]. Together, the results suggest previously unexpected plasticity in stem cell maintenance and differentiation in the adult mammalian intestine (Nie, 2015).

    In the adult Drosophila midgut, which is equivalent to the mammalian stomach and small intestine, ISCs are distributed evenly along the basal side of the monolayered epithelium to support repair. The maintenance and regulation of Drosophila midgut ISCs depend on both intrinsic and extrinsic factors. When a midgut ISC divides, it generates a renewed ISC and an enteroblast (EB) that ceases to divide and starts to differentiate. The ISC-EB asymmetry is established by the Delta-Notch signaling, with Delta in the renewed ISC activating Notch signaling in the newly formed neighboring EB . Growth factors such as Wingless/ Wnt, insulin-like peptides, Decapentaplegic/BMP, Hedgehog and ligands for the EGF receptor and JAK-STAT pathways are secreted from surrounding cells and constitute the niche signals that regulate both ISC division and EB differentiation. ISC-intrinsic factors including Myc, Target of Rapamycin (TOR) and Tuberous Sclerosis Complex act to coordinate the growth and division of ISCs. Furthermore, chromatin modifiers such as Osa, Brahma and Scrawny function within ISCs to regulate Delta expression or ISC proliferation (Nie, 2015).

    This study reports the identification of the leucine zipper protein Bunched (Bun) and the adaptor protein myeloid leukemia factor 1 adaptor molecule (Madm) as intrinsic factors for ISC proliferation. A single bun genomic locus generates multiple predicted transcripts that encode 4 long isoforms, BunA, F, G and P, and 5 short isoforms, BunB, C, D, E, H and O. The first identified mammalian homolog of Bun is TGF-β1 stimulated clone-22 (TSC-22). In the mouse genome four different TSC- 22 domain genes also encode multiple short and long isoforms. All isoforms of Bun and TSC-22 contain an approximately 200 amino acids C-terminal domain where the conserved TSC-box and leucine zippers are located. The originally identified TSC-22 is a short isoform and various assays suggest that it suppresses cancer cell proliferation and may function as a transcriptional regulator. Meanwhile, in Drosophila, the long Bun isoforms positively regulate growth, while the short isoforms may antagonize the function of long isoforms. Transgenic fly assays also demonstrate that the long TSC-22 can rescue the bun mutant phenotypes, whereas short isoforms cannot. These results suggest an alternative model that the long Bun isoforms positively regulate proliferation, while the short isoforms may dimerize with and inhibit the functions of long isoforms (Nie, 2015).

    Madm also can promote growth. The long isoform BunA binds to Madm via a conserved motif located in the N- terminus that is not present in the short Bun isoforms. The molecular function of this novel BunA- Madm complex, nonetheless, remains to be elucidated. The results in this report demonstrate that Bun and Madm modulate the Tuberous Sclerosis Complex-target of Rapamycin (TOR)-eIF4E binding protein (4EBP) pathway to regulate the growth and division of ISCs in the adult midgut (Nie, 2015).

    This report shows that Bun and Madm are intrinsically required for ISC growth and division. The results suggest a model that Bun and Madm form a complex in the cytoplasm to promote cellular growth and proliferation. The evidence that support this model includes the observation that transgenic expressed Bun localizes in the cytoplasm of midgut precursor cells, similar to the results from transfection in S2 cells and immune-staining in eye discs. Bun physically and functionally interacts with Madm, which has also been proposed as a cytoplasmic adaptor protein. Adding a nuclear localization signal to Bun reduced the growth promoting ability of Bun. Although there is a possibility this signal peptide changes the functionality in an unpredicted way, the interpretation is favored that Bun normally acts in the cytoplasm and with Madm to regulate the proliferation of ISCs. This is in contrast to mammalian TSC-22, which was reported to function in the nucleus (Nie, 2015).

    The results seem to contradict a previous publication reporting that TSC-22 arrests proliferation during human colon epithelial cell differentiation. However, this apparent contradiction is resolved when the growing evidence for distinct functions for large and small Bun/ TSC-22 isoforms is considered. The Bun/TSC-22 proteins have short and long isoforms that contain the conserved TSC-box and leucine zippers in the C-terminal domain. The prototypical TSC-22 protein, TSC22D1-001, may act as a transcriptional regulator and repress cancer cell proliferation, particularly for blood lineages. Another recent model suggests that in Drosophila the long Bun isoforms interact with Madm and have a growth promoting activity, which is inhibited by the short Bun isoforms. Similarly, the long isoform, TSC22D1-002, enhances proliferation in mouse mammary glands, whereas the short isoform promotes apoptosis. Unpublished result that transgenic expression of BunB also has lower function than BunA in fly intestinal progenitor cells is consistent with this model where large isoforms have a distinct function, namely in growth promotion (Nie, 2015).

    Loss of either Bun or Madm can potently suppress all the growth stimulation by multiple pathways in the midgut as shown in this report. These results are intrepeted to indicate that Bun and Madm do not act specifically in one of the signaling pathways tested but instead function in a fundamental process required for cell growth, such as protein synthesis or protein turnover. It is therefore speculated that Bun and Madm may regulate the TOR pathway. In support of this idea, it was shown that bunRNAi or MadmRNAi efficiently suppresses the Tuberous Sclerosis Complex 2RNAi-induced cell growth and p4EBP phenotypes. A recent study of genetic suppression of TOR complex 1-S6K function in S2 cells also suggests that Bun and Madm can interact with this pathway. Furthermore, proteomic analyses of Bun and Madm interacting proteins in S2 cells have shown interactions with ribosomal proteins and translation initiation factors. Therefore, a model is proposed that Bun and Madm function in the Tuberous Sclerosis Complex-TOR- 4EBP pathway to regulate protein synthesis in ISCs for their growth, which is a prerequisite for ISC proliferation. Suppression of Tuberous Sclerosis Complex mutant cell growth phenotype by bun or Madm RNAi was substantial but not complete. Earlier papers demonstrated that Bun also interacts with Notch and EGF pathway in ovary follicle cells. Therefore by definition Bun and Madm are neither 100% essential nor restricted to the TOR pathway. The genetic data suggest that Bun and Madm work downstream of Tuberous Sclerosis Complex and upstream of 4EBP, but they could also work in parallel to the TOR pathway components (Nie, 2015).

    ISCs with loss of Tuberous Sclerosis Complex function have substantial cell size increase. Meanwhile, the Bun/ Madm overexpression caused increased ISC division but not cell hypertrophy. Both loss of Tuberous Sclerosis Complex and overexpression of Bun/Madm should promote cell growth but the phenotypes at the end are different. It is speculated that the reason is the Bun/Madm overexpressing ISCs are still capable of mitosis, while the Tuberous Sclerosis Complex mutant ISCs do not divide anymore thereby resulting in the very big cells. In Bun and Madm overexpressing mid- guts, the p-H3+ and GFP+ cell count showed a significant increase, indicating increased mitosis. Therefore, an explanation is that Bun and Madm overexpression may increase cell size/cell growth, but when they grow to certain size they divide, resulting in rather normal cell size (Nie, 2015). The knockout of the Madm mammalian homolog, NRBP1, can cause accumulation of the short isoform TSC22D2. Up-regulation of Madm/NRBP1 has been associated with poor clinical outcome and increased growth of prostate cancer. Further analysis based on this model may reveal whether high ratio of long Bun/TSC22 isoforms over short isoforms may associate with high Madm activity and poor clinical outcomes (Nie, 2015).

    The asymmetrically segregating lncRNA cherub is required for transforming stem cells into malignant cells

    Tumor cells display features that are not found in healthy cells. How they become immortal and how their specific features can be exploited to combat tumorigenesis are key questions in tumor biology.This study describes the long non-coding RNA cherub (long non-coding RNA:CR43283) that is critically required for the development of brain tumors in Drosophila but is dispensable for normal development. In mitotic Drosophila neural stem cells, cherub localizes to the cell periphery and segregates into the differentiating daughter cell. During tumorigenesis, de-differentiation of cherub-high cells leads to the formation of tumorigenic stem cells that accumulate abnormally high cherub levels. cherub establishes a molecular link between the RNA-binding proteins Staufen and Syncrip. As Syncrip is part of the molecular machinery specifying temporal identity in neural stem cells, it is proposed that tumor cells proliferate indefinitely, because cherub accumulation no longer allows them to complete their temporal neurogenesis program (Landskron, 2018).

    Throughout the animal kingdom, stem cells supply tissues with specialized cells. They can do this because they have the unique ability to both replicate themselves (an ability termed self-renewal) and to simultaneously generate other daughter cells with a more restricted developmental potential. Besides their role in tissue homeostasis, stem cells have also been linked to tumor formation. They can turn into so-called tumor stem cells that sustain tumor growth indefinitely. The mechanisms that endow tumor stem cells with indefinite proliferation potential are not fully understood (Landskron, 2018).

    Most Drosophila brain tumors originate from the so-called type II neuroblasts (NBIIs). NBIIs divide asymmetrically into a larger cell that retains NB characteristics and a smaller intermediate neural progenitor (INP). Newly formed immature INPs (iINPs) go through a defined set of maturation steps to become transit-amplifying mature INPs (mINPs). After this, a mINP undergoes 3-6 divisions generating one mINP and one ganglion mother cell (GMC) that in turn divides into two terminally differentiating neurons or glial cells (Landskron, 2018).

    During each NBII division, a set of cell fate determinants is segregated into the INP. Among those are the Notch inhibitor Numb and the TRIM-NHL protein Brain tumor (Brat). Loss of these cell fate determinants leads to the generation of ectopic NB-like cells at the expense of differentiated brain cells. Formation of malignant brain tumors has also been observed upon the depletion of downstream factors that normally maintain the INP fate (Landskron, 2018).

    These features make Drosophila a model for the stepwise acquisition of tumor stem cell properties. When numb or brat are inactivated, the smaller NBII progeny fails to establish an INP fate and initially enters a long transient cell cycle arrest. Only after this lag period, the smaller cell regrows to a NB-sized cell that has acquired tumor stem cell properties and that it is therefore refered to as tumor neuroblast (tNB). NBIIs and ectopic tNBs are indistinguishable in terms of markers. Both cell populations are characterized by the expression of self-renewal genes and lack differentiation markers, but nevertheless behave differently. Shortly after entering pupal stages, NBs decrease their cell volumes successively with each NB division before they exit the cell cycle and differentiate. However, tNBs do not shrink during metamorphosis and continue to proliferate even in the adult fly brain. Moreover, in contrast to wild-type brains, the resulting tumor brains can be serially transplanted into host flies for years, indicating the immortality of these tumors (Landskron, 2018).

    Similarly, mammalian homologues of numb and brat have been shown to inhibit tumor growth. Furthermore, the human brat homologue TRIM3 is depleted in 24% of gliomas and NUMB protein levels are markedly reduced in 55% of breast tumor cases. Therefore, results obtained in these Drosophila tumor models are highly relevant (Landskron, 2018).

    This study used the Drosophila brat tumor model to investigate how tNBs differ from their physiological counterparts, the NBIIs. The results indicate that progression towards a malignant state is an intrinsic process in brat tNBs that does not correlate with stepwise acquisition of DNA alterations. Transcriptome profiling of larval NBs identified the previously uncharacterized long non-coding (lnc) RNA cherub as crucial for tumorigenesis, but largely dispensable for NB development. The data show that cherub is the first identified lncRNA to be asymmetrically segregated during mitosis into INPs, where the initial high cherub levels decrease with time. Upon the loss of brat, the smaller cherub-high cell reverts into an ectopic tNBs resulting in tumors with high cortical cherub. Molecularly, cherub facilitates the binding between the RNA-binding protein Staufen and the late temporal identity factor Syp and consequently tethers Syp to the plasma membrane. Depleting cherub in brat tNBs leads to the release of Syp from the cortex into the cytoplasm and represses tumor growth. These data provide insight into how defects in asymmetric cell division can contribute to the acquisition of tumorigenic traits without the need of DNA alterations (Landskron, 2018).

    It is commonly assumed that cancer cells become malignant and gain replicative immortality by acquiring genetic lesions. Surprisingly, however, the current data indicate that brat tumors do not require additional genetic lesions for the transition to an immortal state. This is not a general feature of Drosophila tumors as genomic instability alone can induce tumors in Drosophila epithelial cells and intestinal stem cells. However, the current results are supported by previous experiments demonstrating that defects in genome integrity do not contribute to primary tumor formation in NBs. Similarly, tumors induced by loss of epigenetic regulators in Drosophila wing discs do not display genome instability. In addition, the short time it takes from the inactivation of brat to the formation of a fully penetrant tumor phenotype would most likely be insufficient for the acquisition of tumor-promoting DNA alterations. More likely, the enormous self-renewal capacity and fast cell cycle of Drosophila NBs requires only minor alterations for the adoption of malignant growth. Interestingly, epigenetic tumorigenesis has been described before in humans, where childhood brain tumors only harbor an extremely low mutation rate and very few recurrent DNA alterations. Comparable observations have been made for leukemia. The current results might help to understand mechanisms of epigenetic tumor formation, which are currently unclear in humans (Landskron, 2018).

    cherub is the first lncRNA described to segregate asymmetrically during mitosis. Once cherub is allocated through binding to the RNA-binding protein Staufen into the cytoplasm of INPs, its levels decrease over time. The results show that the inability to segregate cherub into differentiating cells leads to its accumulation in tNBs. The increasing amount of tumor transcriptome data indicates that a vast number of lncRNAs show increased expression levels in various tumor types. Intriguingly, the mammalian homologue of cherub's binding partner Staufen has been also described to asymmetrically localize RNA in dividing neural stem cells. Hence, besides transcriptional upregulation, asymmetric distribution of lncRNAs between sibling cells might play a role in the accumulation of such RNAs in mammalian tumors (Landskron, 2018).

    The data suggest a functional connection between cherub and proteins involved in temporal neural stem cell patterning. This study that tNBs retain the early temporal identity factor Imp even during late larval stages. However, IGF-II mRNA-binding protein (Imp) expression in brat mutants is heterogeneous and only a subpopulation of tNBs maintains young identity (Landskron, 2018).

    Tumor heterogeneity has also been described for pros tumors, where only a subset of tNBs maintains expression of the early temporal factors Imp and Chinmo. Interestingly, it is this subpopulation that drives tumor growth in prospero tumors (Landskron, 2018).

    Consistent with this, genetic experiments show that 'rejuvenating' tNBs enhances tumor growth and consequently increases the survival of tumor bearing flies, whereas 'aging' tNBs identity has the opposite effect. Although mammalian counterparts of Imp have not yet being shown to act as temporal identity genes, their upregulated expression has been implied in various cancer types. Therefore, temporal patterning of NBs has an essential role in brain tumor propagation in Drosophila (Landskron, 2018).

    The subset of tNBs that retain early identity in tumors is lost in a cherub mutant background. This suggests that cherub itself might regulate temporal identity. In NBs and tNBs cherub regulates Syp localization by facilitating the binding of Syp to Staufen and thus recruiting it to the cell cortex. In tumors depleted of cherub, Syp localizes mainly to the cytoplasm and is no longer observed at the cortex. As the removal of Syp in tNBs leads to enhanced tumor growth and early lethality, those data suggest that cherub could control temporal NB identity by regulating the subcellular localization of Syp(Landskron, 2018).

    How could cherub regulate the function of Syp? The RNA-binding protein Syp is a translational regulator and has been suggested to control mRNA stability. As mammalian SYNCRIP/hnRNP Q interacts with a lncRNA that suppresses translation, cherub might regulate Syp to inhibit or promote the translation of a subset of target mRNAs. In particular, in NBs Syp acts at two stages in NBs during development: Firstly, approximately 60 hr after larval hatching it represses early temporal NB factors, like Imp. Secondly, at the end of the NB lifespan Syp promotes levels of the differentiation factor prospero to facilitate the NB's final cell cycle exit. As cherub depletion in brat tumors leads to decreased tumor growth, it is possible that cherub inhibits the Syp-dependent repression of the early factor Imp, which this study shows to be required for optimal tumor growth. However, cherub mutant NBIIs do not show altered timing or expression of Imp during development. In accordance, brat tumors show high cortical cherub levels, but only a subset of NBs expresses Imp. Rather than rendering Syp completely inactive, it is suggested that cherub decreases the ability of Syp to promote factors important to restrict NB proliferation. As prospero is not expressed in NBIIs, it remains to be investigated which Syp targets are affected by cherub (Landskron, 2018).

    Remarkably, cherub mutants are viable, fertile and do not affect NBII lineages. Neurons generated by NBIIs predominantly integrate into the adult brain structure termed central complex, which is important for locomotor activity. As cherub mutants show normal geotaxis, function of the lncRNA seems dispensable for NBIIs to generate their neural descendants (Landskron, 2018).

    Nevertheless, the conserved secondary RNA structures of cherub and its conserved expression pattern in other Drosophila species suggest that it has a functional role. There are several possibilities why no phenotype is observed upon the loss of cherub. In wild-type flies cherub might confer robustness. A similar scenario was observed in embryonic NBs, in which Staufen segregates prospero mRNA into GMCs. The failure to segregate prospero mRNA does not result in a phenotype, but it enhances the hypomorphic prospero GMC phenotype. Thus segregation of prospero mRNA serves as support for Prospero protein to induce a GMC fate. Similarly, cherub could act as a backup to reliably establish correct Syp levels in NBIIs and in INPs. Alternatively, cherub might fine-tune the temporal patterning by regulating the cytoplasmic pool of Syp in the NBs. Increasing Syp levels have been suggested to determine distinct temporal windows, in which different INPs and ultimately neurons with various morphologies are sequentially born. Therefore, it cannot be excluded that changes in Syp levels lead to subtle alterations in the number of certain neuron classes produced by NBIIs that only reveal themselves in pathological conditions like tumorigenesis (Landskron, 2018).

    This study illustrates how a lncRNA can control the subcellular localization of temporal factors. In addition to temporal NB identity, Syp regulates synaptic transmission and maternal RNA localization. While cherub is not expressed in ovaries or adult heads, Staufen has been implicated in these processes, suggesting that other RNAs might act similarly to cherub. Interestingly, the mammalian Syp homolog hnRNP Q binds the noncoding RNA BC200, whose upregulation is used as a biomarker in ovarian, esophageal, breast and brain cancer. In the future, it will be interesting to investigate whether the mechanism identified in Drosophila is involved in mammalian tumorigenesis as well (Landskron, 2018).

    Drosophila tumor suppressor gene prevents tonic TNF signaling through receptor N-glycosylation

    Drosophila tumor suppressor genes have revealed molecular pathways that control tissue growth, but mechanisms that regulate mitogenic signaling are far from understood. This study reports that the Drosophila TSG tumorous imaginal discs (tid), whose phenotypes were previously attributed to mutations in a DnaJ-like chaperone, are in fact driven by the loss of the N-linked glycosylation pathway component ALG3. tid/alg3 imaginal discs display tissue growth and architecture defects that share characteristics of both neoplastic and hyperplastic mutants. Tumorous growth is driven by inhibited Hippo signaling, induced by excess Jun N-terminal kinase (JNK) activity. Ectopic JNK activation is caused by aberrant glycosylation of a single protein, the fly tumor necrosis factor (TNF) receptor homolog, Grindelwald, which results in increased binding to the continually circulating TNF. These results suggest that N-linked glycosylation sets the threshold of TNF receptor signaling by modifying ligand-receptor interactions and that cells may alter this modification to respond appropriately to physiological cues (de Vreede, 2018).

    Tumorigenesis is ultimately driven by dysregulated cellular signaling that promotes unchecked proliferation. Proliferation-regulating signaling pathways in animals are therefore normally under tight control, to prevent aberrant growth. The primary mechanism of signaling regulation is limited availability of ligand, although levels of receptor can also be regulated, as can receptor availability on the plasma membrane or even its polarized localization. A full understanding of the mechanisms that limit mitogenic signaling is an important goal of both basic biology and cancer research (de Vreede, 2018).

    Major insight into growth regulation has arisen from research in model organisms such as Drosophila melanogaster. For instance, Drosophila studies revealed key steps of receptor tyrosine kinase signaling and uncovered the phenomenon of cell competition. Additional insight into growth regulatory mechanisms has come from the analysis of fly tumor suppressor genes (TSGs). Disruption of a single fly TSG is sufficient to cause overproliferation in epithelial organs of the larva called imaginal discs. Initial genetic screens identified several classes of fly TSGs. The neoplastic TSGs (discs large, lethal giant larvae, and scribble) revealed an intimate link between cell polarity and cell proliferation control, a principle also relevant to human cancers. The hyperplastic TSGs, including hippo, warts, and salvador, uncovered the novel Hippo (Hpo) signal transduction pathway, which is now recognized as a conserved growth control mechanism. Even less prominent Drosophila TSGs such as lethal giant discs have demonstrated important biological concepts (de Vreede, 2018).

    One classic Drosophila TSG that remains understudied is tumorous imaginal discs (tid). Imaginal discs of tid homozygous larvae develop into overgrown masses. Genetic mapping and cytogenetic analyses attributed this phenotype to loss of a conserved molecular chaperone of the DnaJ family. Evidence for a tumor-suppressive role for a mammalian homolog, hTid-1, has been presented. However, the exact molecular mechanism through which tid could regulate cell and tissue proliferation remains mysteriou (de Vreede, 2018 and references therein).

    This study reports that the tid gene was cloned incorrectly. Aberrant cell proliferation in the Drosophila mutant arises not from disruptions to the DnaJ homolog but rather to an adjacent gene that encodes the mannosyltransferase ALG3, involved in N-linked glycosylation. Overgrowth in tid/ALG3 mutants is caused by mis-glycosylation of a single transmembrane protein, the Drosophila tumor necrosis factor (TNF) receptor homolog Grindelwald, which results in downstream activation of Jun N-terminal kinase (JNK) and inactivation of the growth-suppressing Hpo pathway. The results suggest that this post-translational modification modulates ligand-receptor affinity in the TNF receptor (TNFR) pathway and thus provides a regulatory mechanism setting a dynamic threshold for JNK-mediated stress signaling and growth control (de Vreede, 2018).

    This study has shown that mutations in the classic Drosophila TSG tumorous imaginal discs (tid) disrupt the ALG3 homolog CG4084, altering the lipid-linked biosynthetic pathway that generates oligosaccharides for protein N-linked glycosylation. Although altered glycosylation affects many proteins and can induce a unfolded protein response (UPR), this study finds that the growth control phenotype of Alg3 can be ascribed to a single target and a single mechanism. This target is the Drosophila TNFR homolog, whose proper modification at a single extracellular site is required to prevent inappropriate TNF binding, subsequent JNK activation, and downstream Yki-driven overproliferation. It is postulated that N-glycosylation can act as a mechanism to modulate JNK signaling in response to cellular stresses (de Vreede, 2018).

    The alg3 mutations were originally identified for their overgrowth phenotype in imaginal discs. Like most other Drosophila TSGs, this phenotype is caused by changes in Hpo-regulated Yki activation, but alg3 mutants differ in both upstream regulation and downstream targets. Mutations in core Hpo signaling components result in rapid proliferation of disc cells, while the slow growth of alg3 mutant tissue resembles that of the neoplastic TSGs. Nonetheless, the STAT pathway, which is a major mitogenic effector in neoplastic mutants, is not elevated in alg3 tissue. Upstream, JNK-dependent Yki activity is seen in both alg3 and neoplastic mutants. However, JNK activation in neoplastic mutants has been suggested to occur either through ligand-independent Grnd activation caused by alteration to apicobasal polarity or through Grnd-independent mechanisms. In alg3 mutants, polarity is intact and overgrowth entirely relies on a Grnd-Egr axis, specifically the increased sensitivity of misglycosylated Grnd for endocrine Egr. Thus, TNFR signaling induced by altered N-glycosylation seems to define distinct consequences for downstream Hpo-mediated growth control (de Vreede, 2018).

    While this study has not tested biochemical affinities directly, the data are consistent with a model where TNF-binding properties are directly regulated by glycosylation of TNFR. Partial or complete removal of the glycan at N63, within the ligand-binding domain of Grnd, leads to an increase of bound Egr, indicating that N-glycosylation normally limits Grnd engagement and downstream signaling. In Drosophila larvae, Egr is continuously transcribed in the fat body for secretion into the hemolymph, bathing Grnd-expressing tissues, including imaginal discs and IPCs in ligand. The results suggest that proper N-glycosylation of Grnd sets a threshold that prevents tonic signaling in these and other tissues under normal circumstances. This raises the intriguing possibility that cell-autonomous changes in N-glycosylation, perhaps induced by stress inputs, could modulate ligand affinity, allowing a rapid and local response to this endocrine signal under different physiological conditions (de Vreede, 2018).

    The modulation of Grnd ligand binding suggested here echoes the regulation of Notch by the glycosyltransferase Fringe. However, the obligate role of Alg3 in all N-glycan synthesis is fundamentally distinct from Fringe's substrate-specific elaboration of a particular O-glycan. In the case of Notch, the specific sugar residues added by Fringe alter receptor selectivity for one ligand over another. Since either aberrant or absent Grnd N-glycosylation results in increased ligand binding and ectopic signaling, evidence for specific glycan structures in modulating the ligand-receptor interface does not currently exist. Whether the glycan could provide a simple steric obstacle to ligand binding or may regulate it through more complex interactions will await structural studies (de Vreede, 2018).

    Grnd shows strong homology to vertebrate TNFR family members in its extracellular TNF-binding domain, although downstream signaling in the fly acts mainly through JNK, in contrast to mammalian homologs that also signal through nuclear factor κB (NF-κB), p38, and caspases. Among the 29 mammalian TNFR superfamily members, at least seven have predicted N-glycosylation sites in their extracellular domains. Several of these sites have been studied, and their proposed roles vary from promoting signaling to inhibiting it or being functionally neutral. The current results motivate analyses of the receptors BCMA and DR4, which are closely related to Grnd and whose predicted N-glycosylation sites each lie in an analogous location within the ligand-binding domain (de Vreede, 2018 and references therein).

    The data presented above, which highlight a new mechanism for restraining TNF signaling, hint at pathogenic mechanisms for several human diseases. Altered glycosylation is emerging as a frequent hallmark of cancer, in which JNK signaling is increasingly implicated. Moreover, mutations in the extracellular domain of human TNFR1, including predicted N-glycosylation sites, can cause the autoinflammatory disease TRAPS (TNFR-associated periodic syndrome). Because the erroneous activation of Grnd in alg3 mutants is akin to an autoinflammatory response, defective N-glycosylation could be an additional mechanism for hyperactive TNFR1 signaling. Finally, mutations in N-glycosylation pathway enzymes including Alg3 result in recessive genetic diseases called type I congenital disorders of glycosylation (CDG-I). CDG patients exhibit a variety of poorly characterized symptoms associated with multiple organs, and the etiology of CDG is largely unknown. The finding of altered inflammatory TNFR/JNK signaling in analogous fly mutants provides a new avenue to investigate (de Vreede, 2018).

    Warburg effect metabolism drives neoplasia in a Drosophila genetic model of epithelial cancer

    Cancers develop in a complex mutational landscape. Genetic models of tumor formation have been used to explore how combinations of mutations cooperate to promote tumor formation in vivo. This study identified lactate dehydrogenase (LDH), a key enzyme in Warburg effect metabolism, as a cooperating factor that is both necessary and sufficient for epidermal growth factor receptor (EGFR)-driven epithelial neoplasia and metastasis in a Drosophila model. LDH is upregulated during the transition from hyperplasia to neoplasia, and neoplasia is prevented by LDH depletion. Elevated LDH is sufficient to drive this transition. Notably, genetic alterations that increase glucose flux, or a high-sugar diet, are also sufficient to promote EGFR-driven neoplasia, and this depends on LDH activity. This study provides evidence that increased LDHA expression promotes a transformed phenotype in a human primary breast cell culture model. Furthermore, analysis of publically available cancer data showed evidence of synergy between elevated EGFR and LDHA activity linked to poor clinical outcome in a number of human cancers. Altered metabolism has generally been assumed to be an enabling feature that accelerates cancer cell proliferation. These findings provide evidence that sugar metabolism may have a more profound role in driving neoplasia than previously appreciated (Eichenlaub, 2018).

    Cancers develop in a complex mutational landscape. Individual tumors carry hundreds, even thousands, of mutations. Specific tumor types have identifiable signatures, consisting of a small number of relatively common 'driver' mutations. The mutational spectrum can vary in different regions of any given tumor, indicating clonal heterogeneity. This heterogeneity poses a challenge to identify which among the many mutational changes contribute to disease (Eichenlaub, 2018).

    Genetic models of tumor formation have been used to explore how combinations of mutations can cooperate to promote neoplasia. Excess epidermal growth factor (EGF) receptor activity is causally linked to many epithelial cancers, including breast cancer. In Drosophila tumor models, EGF receptor (EGFR) overexpression drives hyperplastic growth, but the tissue does not normally progress to neoplasia. When combined with additional genetic alterations, the hyperplastic imaginal disc tissues can undergo neoplastic transformation and metastasis. Interestingly, specific genetic combinations produce tumors with different phenotypic characteristics, suggesting that these models may provide the means to explore specific cancer phenotypes (Eichenlaub, 2018).

    A growing body of evidence has suggested an association between altered sugar metabolism and cancer risk. In cancer cells, glucose metabolism shifts away from using pyruvate to feed oxidative phosphorylation toward use of lactate in aerobic glycolysis (the Warburg effect). The lactate dehydrogenase enzyme plays a key role in the shift to Warburg metabolism. Altered metabolism is thought to enhance the growth potential of cancer cells by diverting glucose to produce building blocks for increased biomass in the form of amino acids, at the expense of efficiency in ATP production via the tricarboxylic acid (TCA) cycle. Depletion of lactate dehydrogenase (LDH) can reduce tumorigenesis in EGFR (Neu)-dependent breast cancer as well as c-Myc-mediated transformation, indicating an important role for this metabolic shift. LDH was found to be upregulated in a Drosophila tumor model driven by overexpressing the activated vascular endothelial growth factor (VEGF) or platelet-derived growth factor (PDGF) receptor, Pvr, but its contribution to tumor formation was not assessed. This report has identified LDH as a cooperating factor that is both necessary and sufficient for EGFR-driven epithelial neoplasia in vivo. Genetic alterations that increase glucose flux, or a high-sugar diet, were sufficient to promote EGFR-driven neoplasia, and this depends on LDH. These findings provide evidence that Warburg effect metabolism may have a more fundamental role in driving neoplasia than previously appreciated (Eichenlaub, 2018).

    This study shows that LDH overexpression is sufficient to drive neoplasia in combination with EGFR expression. Overexpression of LDHA in a human primary breast cancer cell model promoted a more transformed cellular phenotype. The possible significance of synergy between high LDH in a background of high EGFR activity in human cancer is supported by analysis of the cancer genome atlas (TCGA) datasets: evidence that patients with higher LDHA activity and higher EGFR activity show earlier disease progression in breast cancer, sarcoma, and lower grade gliomas. These effects were only seen when the two factors occurred together, suggesting synergy between EGFR activity and the metabolic shift toward aerobic glycolysis (Eichenlaub, 2018).

    Another study has reported that increases in LDHA were able to promote EMT and invasiveness in renal clear cell carcinoma (ccRCC) and that blocking LDH activity could suppress these phenotypes as well as metastasis of ccRCC in xenografts. Although no evidence was found for an effect of LDHA alone or of LDHA/EGFR synergy in the TCGA ccRCC data, these findings merit further attention (Eichenlaub, 2018).

    The observations reported in this study provide evidence that increased sugar flux, whether dietary or due to increased absorption, can promote neoplastic transformation of EGFR-expressing epithelial tissue. The underlying metabolic changes appear to elicit these effects via the lactate shunt, because the effects of high sugar were abrogated by lowering the level of LDH expression in the tissue. A number of recent studies have begun to link elevated sugar flux to the metastatic phenotype. Together with the current findings, these studies may provide a molecular framework to better understand the links between diet, obesity, and cancer and may help to select patient populations who might benefit from future therapeutic agents targeting lactate dehydrogenase activity (Eichenlaub, 2018).

    The ligand Sas and its receptor PTP10D drive tumour-suppressive cell competition

    Normal epithelial cells often exert anti-tumour effects against nearby oncogenic cells. In the Drosophila imaginal epithelium, clones of oncogenic cells with loss-of-function mutations in the apico-basal polarity genes scribble or discs large are actively eliminated by cell competition when surrounded by wild-type cells. Although c-Jun N-terminal kinase (JNK) signalling plays a crucial role in this cell elimination, the initial event, which occurs at the interface between normal cells and polarity-deficient cells, has not previously been identified. Through a genetic screen in Drosophila, this study identifies the ligand Sas and the receptor-type tyrosine phosphatase PTP10D as the cell-surface ligand-receptor system that drives tumour-suppressive cell competition. At the interface between the wild-type 'winner' and the polarity-deficient 'loser' clones, winner cells relocalize Sas to the lateral cell surface, whereas loser cells relocalize PTP10D there. This leads to the trans-activation of Sas-PTP10D signalling in loser cells, which restrains EGFR signalling and thereby enables elevated JNK signalling in loser cells, triggering cell elimination. In the absence of Sas-PTP10D, elevated EGFR signalling in loser cells switches the role of JNK from pro-apoptotic to pro-proliferative by inactivating the Hippo pathway, thereby driving the overgrowth of polarity-deficient cells. These findings uncover the mechanism by which normal epithelial cells recognize oncogenic polarity-deficient neighbours to drive cell competition (Yamamoto, 2017).

    Normal epithelial cells possess an intrinsic tumour-suppression mechanism against oncogenic neighbours. For instance, in canine kidney cell cultures and zebrafish embryos, oncogenic cells that activate Ras or Src are eliminated from an epithelial monolayer when surrounded by normal cells. Similarly, in the Drosophila imaginal epithelium, oncogenic polarity-deficient cells mutant for scribble (scrib) or discs large (dlg1; hereafter dlg) are eliminated from the tissue when surrounded by wild-type cells. The removal of these surrounding wild-type cells abolishes cell elimination and allows scrib- loss-of-function mutant cells to overproliferate; this context-dependent cell elimination is therefore considered to be cell competition. Genetic studies in Drosophila have revealed that this tumour-suppressive cell competition is driven by JNK-dependent cell death, triggered by the Drosophila tumour necrosis factor (TNF) Eiger. However, the initial mechanism by which normal epithelial cells recognize nearby polarity-deficient cells to drive cell competition have remained unknown (Yamamoto, 2017).

    To explore the initial event, which occurs at the interface between normal cells and oncogenic polarity-deficient cells, an ethyl methanesulfonate (EMS)-based genetic screen was conducted in Drosophila for genes required for wild-type 'winners' to eliminate neighbouring polarity-deficient 'losers'. In the eye imaginal epithelium, clones of homozygous mutant scrib-/- are eliminated when surrounded by wild-type tissue. The elimination of scrib-/- clones is also evident in adult eyes. Using the FLP/FRT-mediated genetic mosaic technique, EMS-induced homozygous mutations were induced only in wild-type winners and screened for mutations that caused an elimination-defective (eld) phenotype in neighbouring scrib- losers. Among 7,490 mutant strains generated, four elimination-defective mutants (eld-4, eld-6, eld-7, and eld-8) that fell into the same lethal complementation group were generated. Clones of scrib- cells surrounded by eld-4 clones were no longer eliminated but instead grew robustly in the eye disc and survived into adult tissue, causing a characteristic melanization phenotype. Notably, clones of eld-4, eld-6, eld-7, or eld-8 cells showed neither a growth disadvantage of their own nor a suppressive effect on the growth of neighbouring wild-type tissue. Thus, the complementation group eld-4/6/7/8 possesses mutations in a gene required for elimination of neighbouring scrib- clones (Yamamoto, 2017).

    Using a series of chromosomal-deficiency lines and subsequent cDNA sequencing, a nonsense mutation in the coding region of the gene stranded at second (sas) was identified in the eld-4 mutant strain. Encoded by sas is a cell-surface ligand protein that has two extracellular domains-von Willebrand factor type C (VWC) and fibronectin type 3 (FN3) domains-as well as a transmembrane domain. Sas is required for proper axon guidance in the nervous system, but its physiological role in epithelia is unknown. Expression of Sas was indeed lost in eld-4 clones, but ectopic expression of Sas within eld-4 clones surrounding scrib-/- clones reversed the elimination-defective phenotype. Moreover, the knockdown of Sas in cells surrounding scrib-/- clones phenocopied the elimination-defective phenotype; a similar elimination-defective phenotype also occurred upon Sas knockdown in cells surrounding dlg-/- mutant eye-disc clones. These data reveal that the cell-surface ligand Sas is required for normal epithelial cells to eliminate neighbouring polarity-deficient cells (Yamamoto, 2017).

    Next, attempts were made to understand the mechanism by which Sas drives the elimination of nearby cells. Sas is normally localized at the apical surface of epithelial cells. Notably, however, this study found that Sas relocalized to the lateral cell surface specifically at the interface between wild-type and scrib-/- or dlg-/- clones. This relocalization of Sas at the clone interface was also observed between wild-type and scrib-/- sas-/- double-mutant clones, indicating that the Sas protein that accumulates at the clone interface is derived from surrounding wild-type cells (Yamamoto, 2017).

    The fact that normal epithelial cells relocalize Sas laterally to eliminate neighbouring oncogenic cells suggests that normal cells transmit a signal to these cells through a cell-surface receptor for Sas. Attempts were made to identify the Sas receptor expressed in polarity-deficient cells. It has been reported that PTP10D, a receptor-type tyrosine phosphatase (RPTP), interacts and functions with Sas during longitudinal axon guidance in the Drosophila nervous system and that Sas-PTP10D trans-signalling occurs through glial-neuronal communication. It was therefore assumed that PTP10D and/or other RPTPs were strong candidates for the Sas receptor in the imaginal epithelium. Given that two extracellular domains of Sas, VWC and FN3, can form homophilic interactions with the same domains of other proteins and that FN3 is a domain commonly shared by RPTPs, Thirty-two RNA interference (RNAi) fly strains were screened that target expression of Drosophila transmembrane proteins bearing either VWC or FN3 domains. Only one RNAi line targeting PTP10D phenocopied the severe elimination-defective and melanization phenotypes when expressed within scrib-/- or dlg-/- mutant clones. Like Sas, PTP10D was relocalized to the interface between scrib-/- and wild-type clones, whereas it normally localized at the apical surface of epithelial cells. This lateral accumulation of PTP10D was almost eliminated when PTP10D-RNAi was expressed within scrib-/- clones, indicating that the PTP10D accumulating at the clone interface derives from scrib-/- mutant cells. Furthermore, immunostaining analysis of scrib-/-sas-/- double-mutant clones indicated that Sas and PTP10D are localized adjacent to each other in neighbouring cells. Notably, the lateral relocalization of Sas and PTP10D at the clone interface was also observed for the neoplastic non-functional tumour-suppressor mutants vps25-/-, erupted-/-, or Rab5DN-expressing cells, all of which are eliminated as losers of cell competition when surrounded by wild-type cells; however, such relocalization was not observed for non-neoplastic polarity stardust-/- or crumbs-/- mutants. These data suggest that in response to the emergence of neoplastic polarity-deficient cells, adjacent normal cells relocalize Sas laterally whereas nearby polarity-deficient cells relocalize PTP10D laterally, thereby driving elimination of polarity-deficient cells through trans-activated Sas-PTP10D signalling (Yamamoto, 2017).

    Next the mechanism by which Sas-PTP10D signalling drives elimination of polarity-deficient cells was investigated. It has previously been shown that the activation of Eiger-JNK signalling in polarity-deficient cells is essential for their elimination. Therefore, a possible mechanism by which PTP10D knockdown in scrib-/- clones results in an elimination-defective phenotype is through inhibition of JNK signalling. However, JNK signalling was still strongly activated in scrib-/- clones expressing PTP10D-RNAi, as assessed by the JNK target MMP1. This indicates that loss of PTP10D drives one or more intracellular signalling events that cause an elimination-defective phenotype in the presence of JNK activation. A strong candidate for this signalling event is activation of Ras signalling, as JNK is converted from pro-apoptotic to pro-growth in the presence of Ras activation. Notably, it has been reported that PTP10D and its mammalian orthologue PTPRJ (also known as DEP1/CD148/SCC1/RPTPeta) negatively regulate epidermal growth factor receptor (EGFR) signalling by directly dephosphorylating the intracellular tyrosine kinase domain of EGFR. This study found that EGFR normally localizes apically in wild-type cells but relocalizes to the lateral surface together with PTP10D at the boundaries between scrib-/- and wild-type clones. More pertinently, EGFR-Ras signalling was strongly elevated in scrib-/- clones expressing PTP10D-RNAi, as assessed by downregulation of the transcription factor Capicua. Moreover, co-knockdown of EGFR and PTP10D in scrib-/- clones completely reversed the elimination-defective phenotype, with EGFR-RNAi alone having only a slight effect on the growth of normal tissue. Furthermore, expression of a constitutively active form of EGFR or Ras caused overgrowth of scrib-/- clones, while expression of dominant-negative form of Ras in scrib-/-PTP10D-RNAi clones strongly suppressed their growth. Thus, scrib clones in the absence of PTP10D signalling activate both JNK and Ras signalling and overgrow in a manner dependent on EGFR signalling. The co-activation of EGFR-Ras and Eiger-JNK signalling causes hyper-accumulation of intracellular F-actin, thereby inactivating the tumour-suppressor Hippo pathway. Inactivation of the Hippo pathway triggers nuclear translocation and activation of the downstream transcriptional co-activator Yorkie (Yki), which induces upregulation of various pro-growth and anti-apoptotic genes. Indeed, scrib-/- clones expressing PTP10D-RNAi strongly accumulated intracellular F-actin and showed strong upregulation of the Yki target gene expanded (ex), as well as an increased nuclear signal of Yki protein; however, scrib mutation alone only slightly upregulated F-actin and ex expression. Furthermore, inhibition of Yki activity by the Yki kinase Warts (Wts) or Yki-RNAi significantly suppressed growth of scrib-/- clones in the absence of PTP10D, while Wts-overexpression or Yki-RNAi alone had little effect on tissue growth. Similar upregulations of EGFR signalling and Yki activity were observed in scrib-/- clones when surrounded by sas-/- eld-4 clones. Finally, he number of dying cells at the boundaries between scrib-/- and wild-type clones was found to be significantly reduced by PTP10D-knockdown, whereas cell proliferation was significantly increased in scrib-/- clones expressing PTP10D-RNAi. Together, these data indicate that when neoplastic polarity-deficient cells emerge in the epithelium, neighbouring non-neoplastic cells restrain EGFR signalling of nearby polarity-deficient cells through a Sas-PTP10D trans-interaction, which enables JNK signalling activated in polarity-deficient cells to drive cell elimination. In the absence of Sas-PTP10D, elevated EGFR-Ras signalling in polarity-deficient cells cooperates with JNK signalling to cause Yki activation, thereby leading to an elimination defect and overgrowth of polarity-deficient cells (Yamamoto, 2017).

    These data indicate that in response to the emergence of oncogenic polarity-deficient cells, Sas and PTP10D relocalize specifically at the clone interface to the respective lateral surfaces of normal or polarity-deficient cells, enabling the ligand and receptor to interact with each other in trans. Thus, Sas-PTP10D acts as a fail-safe system for epithelial tissue, a system that protects against neoplastic development and is normally latent but activates upon oncogenic cell emergence. Notably, the Sas-PTP10D system was not required for other types of cell competition triggered by Minute, Mahjong, Myc or Yki. Although the mechanism by which Sas and PTP10D relocalize to the clone interface is currently unknown, this study found that the apical proteins Bazooka, Patj, and aPKC and the sub-apical protein E-cadherin also relocalize to the lateral surface of the clone boundary. This suggests that the apical cell surface expands to the lateral region at the clone boundary, meaning that Sas and PTP10D meet each other in trans at the clone interface (Yamamoto, 2017).

    The genetic data reveal that Sas and PTP10D act together as tumour suppressors during cell competition. Previous studies have reported that PTPRJ, the mammalian homologue of PTP10D, also acts as a tumour suppressor and negatively regulates EGFR signalling. Although no obvious homologues of Sas have been identified in mammals, thrombospondin-1 and syndecan-2 have been reported to act as ligands for PTPRJ. Given that elimination of scrib-deficient cells by cell competition also occurs in mammalian systems, and that the signalling mechanisms identified in Drosophila are evolutionarily conserved, similar cell-cell recognition mechanisms may help to safeguard human tissues against tumorigenesis (Yamamoto, 2017).

    Structural basis for the activation of the deubiquitinase Calypso by the Polycomb protein ASX

    Ubiquitin C-terminal hydrolase deubiquitinase BAP1 is an essential tumor suppressor involved in cell growth control, DNA damage response, and transcriptional regulation. As part of the Polycomb repression machinery, BAP1 is activated by the deubiquitinase adaptor domain of ASXL1 mediating gene repression by cleaving ubiquitin (Ub) from histone H2A in nucleosomes. The molecular mechanism of BAP1 activation by ASXL1 remains elusive, as no structures are available for either BAP1 or ASXL1. This study presents the crystal structure of the BAP1 ortholog from Drosophila melanogaster, named Calypso, bound to its activator, ASX, homolog of ASXL1. Based on comparative structural and functional analysis, a model for Ub binding by Calypso/ASX, uncover decisive structural elements responsible for ASX-mediated Calypso activation, and characterize the interaction with ubiquitinated nucleosomes. The results give molecular insight into Calypso function and its regulation by ASX and provide the opportunity for the rational design of mechanism-based therapeutics to treat human BAP1/ASXL1-related tumors (De, 2018).

    Mutations in the Drosophila tricellular junction protein M6 synergize with Ras(V12) to induce apical cell delamination and invasion

    Complications from metastasis are responsible for the majority of cancer-related deaths. Despite the outsized medical impact of metastasis, remarkably little is known about one of the key early steps of metastasis: departure of a tumor cell from its originating tissue. It is well documented that cellular delamination in the basal direction can induce invasive behaviors, but it remains unknown if apical cell delamination can induce migration and invasion in a cancer context. To explore this feature of cancer progression, a genetic screen was performed in Drosophila, and mutations in the protein M6 were found to synergize with oncogenic Ras to drive invasion following apical delamination without crossing a basement membrane. Mechanistically, it was observed that M6-deficient Ras(V12) clones delaminate as a result of alterations in a Canoe-RhoA-myosin II axis that is necessary for both the delamination and invasion phenotypes. To uncover the cellular roles of M6, this study showed that it localizes to tricellular junctions in epithelial tissues where it is necessary for the structural integrity of multicellular contacts. This work provides evidence that apical delamination can precede invasion and highlights the important role that tricellular junction integrity can play in this process (Dunn, 2018).

    Cells are known to delaminate from their tissues in both the apical and basal directions during development and in disease conditions. Importantly, cell delamination plays a vital role in cancer progression as it is one way that a cancer cell can escape its originating tissue before spreading to more distant sites. During tumor progression, different models have revealed that cell delamination in the apical direction can lead either to the elimination of the delaminated cells or to the overgrowth of those cells. However, invasive behaviors have not been observed to follow apical delamination but instead have been shown to occur only through basal delamination (Dunn, 2018).

    During basal delamination-induced invasion, basement membrane degradation and cell invasion into the underlying tissue can be observed in fixed tissues. On the other hand, if cancer cells leave the tissue by migrating and invading following apical delamination, the invasion would not leave such a histologically visible trail as this invasion could occur without crossing the basement membrane but instead through migration along connected tissues. As such, alternate methods in a suitable system would be needed to recognize if apical delamination is able to induce invasion. Thus, although previous work has documented direct basal delamination and invasion during metastasis in animal models and human patients, it does not preclude the possibility that invasion can be initiated by apical delamination as well. Drosophila cancer models are well suited to address the role of apical delamination in inducing invasive behaviors due to their simple tissue architecture that allows for the easy identification of an apical delamination event, as well as established techniques to image intact living tissues over time to follow the fates of apically delaminated cells. This study documents that cell migration and invasion can be induced via apical delamination through the characterization of a tumor suppressor, M6, in Drosophila (Dunn, 2018).

    While bicellular junctions have been well studied for their roles in tissue integrity and signaling, the importance of TCJs has been gradually coming to light in recent years as they have been shown to be key players in ionic barrier formation and maintenance, pathogen spread, and orientation of cell division. This study demonstrates that inactivation of a TCJ protein, M6, disrupts the structural integrity of multicellular contacts and induces apical delamination and invasion of otherwise benign RasV12 tumors in a manner dependent upon a Cno-RhoA-MyoII axis. This study thus provides a causative role for TCJ mutations in driving delamination and invasion in vivo, highlighting the importance of these junctions in tissue integrity and cancer biology (Dunn, 2018).

    This study demonstrates a functional link between tricellular junctions and RhoA, which is a known cytoskeletal regulator. This finding adds to recent work that has begun suggesting that TCJs act as centers for cytoskeletal organization. It will be interesting to further learn the mechanisms and consequences of functionally linking RhoA and cytoskeletal components to TCJs. Additionally, RhoA is known to affect a variety of proteins and cellular processes in addition to Sqh. As such, it is highly possible that RhoA is inducing apical delamination and invasion through multiple routes in addition to its effects on sqh. Also, since Cno localizes at the adherens junctions, which are apical to M6, it is plausible that M6 only indirectly affects Cno, and thus RhoA, through alterations in epithelial integrity rather than through direct means (Dunn, 2018).

    Finally, invasion of cancer cells into surrounding tissues was previously thought to occur only through direct basal delamination and subsequent invasion. This work shows that apical delamination can also precede migration and invasion to distant tissues. Furthermore, since no basement membrane degradation was observed in invasive RasV12; M6-/- clones, the invasion most likely occurs along connected tissues rather than through an apical delamination to the basal penetration route, but further experiments are needed to confirm this hypothesis. Although mammalian anatomy differs markedly from that of the simple architecture of the Drosophila imaginal discs, it will be interesting to learn if apical delamination, such as is observed in early stage human breast cancer, can also precede invasion in mammalian models. Further investigation into this paradigm of apical delamination-induced invasion could aid in understanding of the mechanisms underlying cancer progression and metastasis (Dunn, 2018).

    Dissemination of Ras(V12)-transformed cells requires the mechanosensitive channel Piezo

    Dissemination of transformed cells is a key process in metastasis. Despite its importance, how transformed cells disseminate from an intact tissue and enter the circulation is poorly understood. This study used a fully developed tissue, Drosophila midgut and describes the morphologically distinct steps and the cellular events occurring over the course of Ras(V12)-transformed cell dissemination. Notably, Ras(V12)-transformed cells formed the Actin- and Cortactin-rich invasive protrusions that were important for breaching the extracellular matrix (ECM) and visceral muscle. Furthermore, the essential roles were uncovered of the mechanosensory channel Piezo in orchestrating dissemination of Ras(V12)-transformed cells. Collectively, our study establishes an in vivo model for studying how transformed cells migrate out from a complex tissue and provides unique insights into the roles of Piezo in invasive cell behavior (Lee, 2020).

    Evidence for a novel function of Awd in maintenance of genomic stability

    The abnormal wing discs (awd) gene encodes the Drosophila homolog of NME1/NME2 metastasis suppressor genes. Awd acts in multiple tissues where its function is critical in establishing and maintaining epithelial integrity. This study analysed awd gene function in Drosophila epithelial cells using transgene-mediated RNA interference and genetic mosaic analysis. awd knockdown in larval wing disc epithelium leads to chromosomal instability (CIN) and induces apoptosis mediated by activation of c-Jun N-terminal kinase. Forced maintenance of Awd depleted cells, by expressing the cell death inhibitor p35, downregulates atypical protein kinase C and DE-Cadherin. Consistent with their loss of cell polarity and enhanced level of matrix metalloproteinase 1, cells delaminate from wing disc epithelium. Furthermore, the DNA content profile of these cells indicates that they are aneuploid. Overall, these data demonstrate a novel function for awd in maintenance of genomic stability. These results are consistent with other studies reporting that NME1 down-regulation induces CIN in human cell lines and suggest that Drosophila model could be successfully used to study in vivo the impact of NME/Awd induced genomic instability on tumour development and metastasis formation (Romani, 2017).

    Genomic stability is critical for cell survival and development and several cellular mechanisms act to maintain genomic integrity. Failure of these mechanisms underlies aging and can lead to malignancies such as cancer and age-related neurodegenerative diseases. Chromosomal instability (CIN) is a form of genomic instability that often leads to aneuploidy, a deleterious condition characterised by copy number changes affecting part or whole chromosomes. Several dysfunctions could lead to CIN. Defective activity of the spindle assembly checkpoint (SAC), a signalling pathway that blocks anaphase onset in response to mis-attachment of chromosomes to the mitotic spindle, leads to CIN and aneuploidy. Work in Drosophila showed that loss of function of SAC genes as well as loss of function of genes involved in spindle assembly, chromatin condensation and cytokinesis induce CIN. More recent work in larval disc epithelia has shown that down-regulation of these genes causes apoptotic cell death trough activation of the c-Jun N-terminal kinase (JNK) pathway. Interestingly, blocking CIN-induced apoptotic cell death induces tumourigenic behaviour including basement membrane degradation, cell delamination, tissue overgrowth and aneuploidy (Romani, 2017).

    The abnormal wing discs (awd) gene encodes the Drosophila homolog of NME1/2 metastasis suppressor genes. Awd is a well-known endocytic mediator whose function is required in multiple tissues during development. Genetic studies showed that Awd endocytic function ensures appropriate internalisation of chemotactic signalling receptors such as platelet-derived growth factor/VEGF receptor (PVR) and fibroblast growth factor receptor (FGFR) and thus it regulates invasion and cellular motility. Furthermore, this endocytic function regulates Notch receptor trafficking and is required for maintenance of epithelial integrity as it controls the turnover of adherens junction components in ovarian somatic follicle cells. Consistent with the high degree of functional conservation between Awd and its mammalian counterparts, recent studies have shown a role for the NME1/2 proteins in vesicular transport (Romani, 2017).

    This work has extended an analysis of the functional conservation between Awd and NME1/2 proteins. Since loss of NME1 gene function in human cell culture leads to polyploidy, this study have explored the role of Awd in maintenance of genomic stability. The data show that knockdown of awd in wing disc cells leads to CIN and to the CIN-induced biological responses mediated through JNK activation. Furthermore, when combined with block of apoptosis, down-regulation of awd leads to cell delamination and aneuploidy. Thus, the results of this in vivo analysis show a novel function for awd in maintenance of genomic stability (Romani, 2017).

    Depletion of Awd triggers JNK-mediated cell death of wing disc cells and blocking the cell death machinery results in aneuploidy and cell delamination without overt hyperproliferative effect. Overgrowth of wing disc hosting aneuploid cells is due to activation of the JNK pathway that promotes expression of Wingless (Wg) upon blocking of apoptotic cell death. Wg is a mitogenic molecule required in the imaginal discs for growth and patterning and its expression in the aneuploid, delaminating CIN cells triggers growth of neighbouring non-delaminating cells. However, awd J2A4 mutant wing disc cells do not express Wg as a consequence of faulty Notch signalling; therefore, these cells cannot promote hyperplasia of the surrounding tissue. Furthermore, lack of hyperproliferation is also observed when an aneuploid condition arises from impaired activity of genes controlling karyokinesis. The diaphanous gene (dia) codes for an actin-regulatory molecule that is required during acto-myosin driven contraction of metaphase furrows. Simultaneous depletion of dia gene expression and blocking of apoptosis do not lead to hyperplastic growth probably due to defective karyokinesis. Intriguingly, Awd is a microtubule-associated nucleoside diphosphate kinase that converts GDP to GTP and the analysis of awd mutant larval brain showed mitotic defects correlated with defective microtubule polymerisation. This raises the possibility that the Awd kinase function plays a role in GTP supply to proteins such as Orbit that are required for stabilisation of spindle microtubules (Romani, 2017).

    Two lines of evidence further support the hypothesis that Awd could be involved in karyokinesis. The first comes from studies showing that endosome trafficking and transport to the intercellular bridges of dividing cells plays a critical role during abscission, the last step of karyokinesis. In addition, remodelling the of plasma membrane that underlies nuclear divisions in the syncytial embryo and cellularisation also requires endocytosis. Embryonic cellularisation requires the dynamin encoded by the shibire (shi) locus and Rab5 GTPase function, since loss of function of either genes arrests ingression of metaphase furrows. Awd functionally interacts with shi locus and Awd is also required for Rab5 function in early endosomes. Thus, a possible role for Awd in cytokinesis should be considered (Romani, 2017).

    The second line of evidence comes from studies on NME1, the human homolog of awd gene. This metastasis suppressor gene shares about 78% of amino acid identity with the awd gene. Down-regulation of NME1 gene expression in diploid cells results in cytokinesis failure and leads to tetraploidy. The in vivo results show that Awd plays a role in maintenance of genomic stability, confirming the high degree of conservation between NME1 and Awd proteins. Drosophila studies have already been crucial in identification of NME1 function in epithelial morphogenesis, and the present work shows that this function can be a useful model for impact on tumour development and progression (Romani, 2017).

    The transcription factor Ets21C drives tumor growth by cooperating with AP-1

    Tumorigenesis is driven by genetic alterations that perturb the signaling networks regulating proliferation or cell death. In order to block tumor growth, one has to precisely know how these signaling pathways function and interplay. This study has identified the transcription factor Ets21C as a pivotal regulator of tumor growth, and a new model is proposed of how Ets21C could affect this process. A depletion of Ets21C strongly suppressed tumor growth while ectopic expression of Ets21C further increased tumor size. It was confirmed that Ets21C expression is regulated by the JNK pathway, and Ets21C was shown to acts via a positive feed-forward mechanism to induce a specific set of target genes that is critical for tumor growth. These genes are known downstream targets of the JNK pathway, and their expression was demonstrated to not only depend on the transcription factor AP-1, but also on Ets21C, suggesting a cooperative transcriptional activation mechanism. Taken together this study shows that Ets21C is a crucial player in regulating the transcriptional program of the JNK pathway and enhances understanding of the mechanisms that govern neoplastic growth (Toggweiler, 2016).

    This study has identified the transcription factor Ets21C as a crucial regulator of tumor growth and demonstrates that its expression is activated by the JNK pathway. Moreover, Ets21C was shown to regulate the expression of specific target genes that induce and sustain growth and invasiveness of RasV12 dlgRNAi tumors, possibly via a cooperation with AP-1 (Toggweiler, 2016).

    The closest human orthologs of Ets21C ETS-related gene (ERG) and Friend leukemia virus-induced erythroleukemia 1 (FLI-1), have also been linked to tumorigenesis. ERG is overexpressed in acute myeloid leukemia (AML) and is associated with a poor prognosis, whereas higher FLI-1 expression has been detected in triple negative breast cancer or in metastatic melanoma. This study shows that Ets21C is not only sufficient to induce tumorigenesis, but required for tumor growth as a depletion of Ets21C strongly reduced tumor size. The smaller tumor size was accompanied by decreased levels of target genes known to drive tumor growth and malignancy, further underscoring the relevance of Ets21C. The finding that an Ets21C loss of function is blocking tumor growth so efficiently, suggests a more fundamental role for Ets21C in tumor growth than previously assumed. Kulshammer (2015) also tested an Ets21CRNAi in RasV12, scrib−/− tumors, but was not able to observe any remarkable effects besides a partial rescue of the pupariation delay that is commonly associated with neoplastic tumor growth in flies. An explanation for this discrepancy might simply be different strengths of the RNAi lines used (Toggweiler, 2016).

    Loss of epithelial polarity caused by a depletion of dlg activates the JNK pathway that critically affects tumor growth. In agreement with previous studies, this study found elevated Ets21C transcript levels in RasV12 dlgRNAi tumors and showed that JNK signaling is required for the upregulation. The JNK pathway activates the AP-1 transcription factor, which in its prototypical form is a dimer consisting of Jun and Fos proteins. In the RasV12, scrib−/− context, Fos has been described as the main effector of the JNK pathway, as a depletion diminishes tumor growth and abrogates induction of target genes, while no such effect have been observed for Jun. However, the current data point towards at least a partial role for Jun activating target genes, since a knockdown of Jun reduced expression of Ets21C and Mmp1 (Toggweiler, 2016).

    Given the strong effects that Ets21C exerts on tumor growth, attempts were made to identify Ets21C dependent target genes that could explain this phenomenon. Transcriptional profiles revealed an upregulation of Mmp1, Pvf1 and upd1 in tumors that overexpressed Ets21C and downregulated in tumors depleted of Ets21C. Mmp1, Pvf1 and upd1 have previously been shown to be induced by the JNK pathway and to be essential for tumor growth and invasion. Besides upd1, also upd2 and upd3 have been reported to be induced in neoplastic tumors in a JNK pathway dependent manner. In agreement with these reports, an induction was observed of upd2 and upd3 in RasV12 dlgRNAi Ets21C tumors, but to a lesser extent than upd1. This might on the one hand depend on the use of dlg instead of scrib a stronger activation of Upd cytokines has been found in scrib mutant wing discs compared to dlg mutants. On the other hand, additional factors might influence transcriptional activation such as the presence of RasV12, the exact timing of sample collection or the genetic system used. Furthermore, the strength of induction might not necessarily correlate with the effect on tumor growth. Although upd3 exhibits the strongest upregulation in neoplastic tumors, it has been shown that co-expression of Upd1 and Upd2 together with RasV12 leads to much larger tumors than the combination of RasV12 and Upd3 (Toggweiler, 2016).

    While Ets21C is able to stimulate the expression of the JNK downstream targets Mmp1, Pvf1 and upd1, no obvious regulation was observed of canonical JNK pathway targets such as the phosphatase puc or the apoptosis inducers hid or rpr, suggesting that Ets21C only regulates a specific set of JNK pathway targets. In contrast, a previous study has described a slight upregulation of puc in RasV12 Ets21C tumors. Trivial differences such as when the samples were collected, transgene strength, or genetic background could account for this difference. For example, an elevation of puc expression could also originate from an indirect increase in JNK activity due to stresses in an older, larger tumor. These results are entirely consistent with the model that Ets21C can activate certain JNK targets in a context dependent manner (Toggweiler, 2016).

    Finally, it was asked if and how Ets21C regulates transcriptional outputs of the JNK pathway. (1) Ets21C could interact with Jun and/or Fos. (2) Ets21C could activate or interact with an unknown factor that feeds back on the JNK pathway. (3) Ets21C could use a combination of both (1) and (2). This study showed genetically that in RasV12 dlgRNAi tumors the effects of Ets21C, both phenotypically and on a target genes level, fully depend on an active JNK pathway. If the latter is blocked, for example, by co-expressing BskDN with RasV12 dlgRNAi and Ets21CHA, tumors remain small and there is no induction of Mmp1, Pvf1 or upd1. These results support all possibilities (1)-(3), but exclude an autonomous function of Ets21C. A physical interaction between Ets21C and Jun and Fos has previously been proposed based on large scale mass-spectroscopy data. This study shows that HA-tagged Ets21C does indeed bind FLAG-tagged Jun or Fos, showing that the proteins could interact physically to regulate target gene expression. The binding is consistent with studies in mammalian systems that have shown a physical interaction between AP-1 and ETS proteins including ERG, the mammalian homolog of Ets21C. In agreement with a cooperative transcriptional activation, Ets21C and AP-1 binding sites were found co-occurring in the putative regulatory regions of the analyzed target genes. It is therefore thought that Ets21C likely activates target genes via a cooperation with AP-1. However, additional factors that are activated by the JNK pathway might contribute to target gene activation as well and could explain why Ets21C activates certain JNK downstream target genes and others not (Toggweiler, 2016).

    In summary, this study shows that Ets21C fulfills a crucial role in the regulation of neoplastic tumor growth as a loss of function critically reduced tumor growth, whereas an excess of Ets21C further increased tumor size. While Ets21C was previously accredited only with a role in fine-tuning the transcriptional program of neoplastic tumors, the current results point towards a more fundamental role as activator of a specific set of target genes that drive tumor growth and invasion (Toggweiler, 2016).

    Cytoneme-mediated signaling essential for tumorigenesis

    Communication between neoplastic cells and cells of their microenvironment is critical to cancer progression. To investigate the role of cytoneme-mediated signaling as a mechanism for distributing growth factor signaling proteins between tumor and tumor-associated cells, EGFR and RET Drosophila tumor models were analyzed, and several genetic loss-of-function conditions were tested that impair cytoneme-mediated signaling. Neuroglian, capricious, Irk2, SCAR, and diaphanous are genes that cytonemes require during normal development. Neuroglian and Capricious are cell adhesion proteins, Irk2 is a potassium channel, and SCAR and Diaphanous are actin-binding proteins, and the only process to which they are known to contribute jointly is cytoneme-mediated signaling. It was observed that diminished function of any one of these genes suppressed tumor growth and increased organism survival. It was also noted that EGFR-expressing tumor discs have abnormally extensive tracheation (respiratory tubes) and ectopically express Branchless (Bnl, a FGF) and FGFR. Bnl is a known inducer of tracheation that signals by a cytoneme-mediated process in other contexts, and it was determined that exogenous over-expression of dominant negative FGFR suppressed tumor growth. These results are consistent with the idea that cytonemes move signaling proteins between tumor and stromal cells and that cytoneme-mediated signaling is required for tumor growth and malignancy (Fereres, 2019).

    A positive feedback loop between Myc and aerobic glycolysis sustains tumor growth in a Drosophila tumor model

    Cancer cells usually exhibit aberrant cell signaling and metabolic reprogramming. However, mechanisms of crosstalk between these processes remain elusive. This study shows that in an in vivo tumor model expressing oncogenic Drosophila Homeodomain-interacting protein kinase (Hipk), tumor cells display elevated aerobic glycolysis. Mechanistically, elevated Hipk drives transcriptional upregulation of Drosophila Myc (dMyc; MYC in vertebrates) likely through convergence of multiple perturbed signaling cascades. dMyc induces robust expression of pfk2 (encoding 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase; PFKFB in vertebrates) among other glycolytic genes. Pfk2 catalyzes the synthesis of fructose-2,6-bisphosphate, which acts as a potent allosteric activator of Phosphofructokinase (Pfk) and thus stimulates glycolysis. Pfk2 and Pfk in turn are required to sustain dMyc protein accumulation post-transcriptionally, establishing a positive feedback loop. Disruption of the loop abrogates tumorous growth. Together, this study demonstrates a reciprocal stimulation of Myc and aerobic glycolysis and identifies the Pfk2-Pfk governed committed step of glycolysis as a metabolic vulnerability during tumorigenesis (Wong, 2019).

    In the 1920s, Otto Warburg first discovered that cancer cells vigorously take up glucose and preferentially produce lactate even in the presence of oxygen, a phenomenon now widely termed the Warburg effect or aerobic glycolysis. Despite his pioneering work, the Warburg effect was largely disregarded for the subsequent decades. After the discoveries of oncogenes and tumor suppressor genes, cancers are generally considered as genetic diseases rather than metabolic ones. Not until the 1980s did the revisiting of the Warburg effect in connection with oncogenes spark substantial research in cancer metabolism, laying the foundation for 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) positron emission tomography (PET) in clinical cancer diagnosis, recognition of metabolic reprogramming as a hallmark of cancer and development of anti-cancer agents targeting aerobic glycolysis (Wong, 2019).

    How the Warburg effect arises and contributes to tumor progression have always been the center in the field of cancer metabolism. Warburg hypothesized that mitochondrial impairment is the cause of aerobic glycolysis and cancer, which has sparked much controversy among scientists. Contrary to his idea, the current, widely-accepted view is that oncogenic drivers such as RAS, MYC, hypoxia-inducible factors (HIFs) and steroid receptor coactivators (SRCs) promote cancer cell proliferation and directly stimulate aerobic glycolysis through regulating transcriptional expression or catalytic activities of metabolic enzymes. Several explanations for the Warburg effect have been put forward, including rapid adenosine triphosphate (ATP) production and de novo biosynthesis of macromolecules. Recently, Warburg effect functions are being re-evaluated as a growing body of evidence shows that metabolic rewiring in cancers impacts cell signaling and epigenetics (Wong, 2019).

    Drosophila has proven to be a powerful genetic model organism for studying tumorigenesis in vivo largely due to high conservation of genes and signaling cascades between human and flies and reduced genetic redundancy. In a recent study, it was shown that elevation of Drosophila Hipk causes an in vivo tumor model characterized by tissue overgrowth, loss of epithelial integrity and invasion-like behaviors (Blaquiere, 2018). The tumorigenic roles of Drosophila Hipk seem to be conserved in mammals as the four members of the HIPK family (HIPK1-4) are also implicated in certain cancers. For example, HIPK1 is highly expressed in breast cancer cell lines, colorectal cancer samples and oncogenically-transformed mouse embryo fibroblasts. Also, HIPK2 is elevated in certain cancers including cervical cancers, pilocytic astrocytomas, colorectal cancer cells and in other proliferative diseases, such as thyroid follicular hyperplasia. To gain a better understanding of cancer metabolism, this study set out to use the fly Hipk tumor model to investigate whether and how cellular metabolism is altered in tumor cells. Hipk-induced tumorous growth was found to be accompanied by elevated aerobic glycolysis. Furthermore, novel feedback mechanisms were identified leading to prolonged dMyc expression and hence tumorigenesis. This study reveals potential metabolic vulnerabilities that could be exploited to suppress tumor growth (Wong, 2019).

    Multiple approaches and tools have been developed to measure glycolysis including measurement of the extracellular acidification rate (ECAR), fluorescent probes, biosensors, fluorescent/colorimetric assays and mass spectrometry. In the Hipk tumor model, the tumor cells are surrounded by wild-type cells. This study primarily used fluorescence imaging such that it was possible to examine changes in metabolite levels, gene and protein expression between tumor cells and the adjacent wild-type cells. This work delineates the causes and significance of metabolic changes in tumor cells (Wong, 2019).

    Drosophila tumor models frequently acquire metabolic changes, especially the Warburg effect. For instance, epidermal growth factor receptor (EGFR)-driven tumors, tumors with activated platelet-derived growth factor (PDGF)/vascular endothelial growth factor (VEGF) receptor (Pvr) and tumors with polarity loss (such as scrib or dlg mutant tumors) feature robust upregulation of Ldh among other glycolytic genes. Depletion of Ldh was shown to reduce growth of EGFR-driven tumors but not tumors with polarity loss. In another study RasV12scrib-/- tumors, elevated glucose uptake was evident, but its significance was not evaluated. Similar to the previously described models, Hipk tumor cells exhibited elevated glucose metabolism. Metabolic reprogramming was driven by dMyc upregulation. Although the transcript levels of another glycolytic inducer sima remained unchanged, it was not possible to eliminate the possibility that Sima protein levels are altered in the tumor cells. Thus, whether Sima is involved in the tumor growth warrants further studies. Genetical inhibition of Pfk or Pfk2 was sufficient to block Hipk-induced tumorigenesis whereas depletion of Pgk, Pyk or Ldh at most slightly reduced tumor growth. Therefore, the data suggest that targeted but not generic inhibition of glycolysis is required to abrogate tumor growth (Wong, 2019).

    A feedback loop between dMyc and aerobic glycolysis as a metabolic vulnerability in tumor cells Although MYC functions in cancer metabolism are widely recognized, MYC is generally considered 'undruggable' largely due to its nuclear localization, lack of an enzymatic 'active site', and indispensable roles during normal development. Hence, limited therapeutic strategies have been developed, and identification of the 'druggable' regulatory proteins of MYC becomes critical (Wong, 2019).

    This study reveals two modes of dysregulation of endogenous dMyc specifically during Hipk-induced tumorigenesis. The first mode is transcriptional stimulation of dMyc likely as a consequence of convergence of multiple signaling outputs. In a previous study, it was found that genetically targeting individual signaling cascades failed to restrain Hipk tumor growth (Blaquiere, 2018). This is likely due to the redundancy of signaling cascades in inducing dMyc upregulation. It is also interesting to note that dMyc upregulation and the associated tumor growth are most striking in the wing hinge. The pouch region, however, seems more refractory to such alterations. The lowest intracellular glucose levels and robust Ldh upregulation were observed in the hinge area. dMyc-dependent glucose uptake, on the other hand, was enhanced in both hinge and pouch regions even though dMyc protein buildup is undetectable in the wing pouch, suggesting that glucose uptake is particularly sensitive to changes in dMyc levels. The data therefore imply that the Warburg effect and the tumor growth phenotype may be linked to the dMyc levels being induced. The region-specific susceptibility to tumorigenic stimuli has been previously described and the hinge region was coined a 'tumor hotspot' because of its unique epithelial cell architectures and high endogenous JAK/STAT signaling. It is possible that such features in the hinge region also contribute to the sensitivity of dMyc upregulation by elevated Hipk and other signaling pathways. Further studies are required to verify this proposition (Wong, 2019).

    The second mode of dMyc regulation is the metabolic control of dMyc protein levels by aerobic glycolysis. Specifically, this study found that the rate-limiting enzymes Pfk2 and Pfk are required to perpetuate dMyc buildup in tumor cells in a post-transcriptional manner. Similar to the effects on tumor growth, while pfk2/pfk knockdown prevented dMyc accumulation, knockdown of other glycolytic enzymes had little effect on sustaining dMyc accumulation. Such a disparity could possibly be explained by the potency of the glycolytic enzymes in controlling glycolytic flux. While Pfk2 and Pfk govern the rate-limiting, committed step of glycolysis, RNAi targeting other glycolytic genes may fail to render the corresponding steps rate-limiting, especially in tumors with elevated aerobic glycolysis. In other words, knockdown of other glycolytic enzymes may not reach the threshold that would restrict glycolytic flux as potently as pfk2/pfk knockdown. Given that Pfk2 is mis-expressed in tumor cells but not in normal cells and that dMyc accumulation is most sensitive to the Pfk2-Pfk mediated committed step of glycolysis specifically in tumor cells, targeting Pfk2 may be a favorable, selective metabolic strategy in the treatment of cancers, especially those displaying ectopic MYC expression (Wong, 2019).

    The functions of mammalian PFK and PFKFB in controlling glycolytic flux are well-defined. The allosteric regulation of PFK by F2,6-BP seems to be conserved among mammals and insects as F2,6-BP is able to activate insect Pfk, likely due to the conserved amino acid residues for F2,6-BP binding. This suggests that Pfk2 can induce Pfk activation and hence glycolytic flux through biosynthesizing F2,6-BP. A previous report shows that flies lacking pfk2 exhibited levels of circulating glucose and reduced sugar tolerance, providing a functional link between Pfk2 and glucose metabolism. This study has provided evidence that both pfk2 and pfk knockdown larvae exhibited significant decreases in pyruvate contents, confirming a conserved role of Pfk2 in regulating glycolytic flux (Wong, 2019).

    Recently, the roles of PFK and PFKFB in the regulation of transcription factors or cofactors have drawn considerable attention. For example, vertebrate PFK was shown to physically interact with TEAD factors to stimulate YAP/TAZ activity, thus promoting proliferation and malignancy in cancer cells. PFKFB4 (an isoform of PFK2) phosphorylates and activates oncogenic SRC-3 to promote breast cancers through stimulating purine synthesis. These studies point to the fact that PFK and PFKFB functions are not restricted to metabolic regulation. Thus, it is tempting to speculate that Pfk2/Pfk may sustain ectopic dMyc through a direct interaction with or even phosphorylation of dMyc. That being said, as knockdown of Pfk2 or Pfk had no effects on dMyc protein levels under normal conditions, it is likely that a tumorigenic environment with active aerobic glycolysis is necessary for the metabolic control of dMyc in particular through the committed step governed by Pfk2/Pfk. In summary, this study shows glycolytic enzymes Pfk and Pfk2, likely through their core biochemical role in stimulating glycolytic flux, glycolysis-independent actions or both, act as key players in coupling metabolic demands with growth signals to achieve cancer progression (Wong, 2019).

    Spz/Toll-6 signal guides organotropic metastasis in Drosophila

    Targeted cell migration plays important roles in developmental biology and disease processes including metastasis. Drosophila tumors exhibit traits characteristic of human cancers, providing a powerful model to study developmental and cancer biology. This study finds that cells derived from Drosophila eye disc tumors also display organ specific metastasis to invade receptive organs but not wing disc. Toll receptors are known to affect innate immunity and tumor inflammatory microenvironment by modulating the NF-kappaB pathway. RNAi screen and genetic analyses show that Toll-6 is required for migration and invasion of the tumor cells. Further, receptive organs express Toll-ligands, Spz family molecules, and ectopic Spz expression renders wing disc receptive to metastasis. Finally, Toll-6 promotes metastasis by activating JNK signaling, a key regulator of cell migration. Hence, this study reports Toll-6 and Spatzle as a new pair of guidance molecules mediating organ-specific metastasis behavior and highlight a novel signaling mechanism for Toll family receptors (Mishra-Gorur, 2019).

    Targeted cell migration and invasion play important roles in a variety of biological and disease processes. This phenomenon is exemplified by organotropic metastasis in cancer progression. Tumor metastasis is the leading cause of death for cancer patients. A fundamental feature of metastasis is the ability of distinctive tumor types to colonize different organ sites, which depends on the inherent properties of the tumor cells and their interaction with the host tissue. However, the mechanism of organotropic metastasis is not well delineated. Deciphering the underlying molecular mechanism(s) of interactions between tumor cells and secondary host tissue is essential for understanding metastasis (Mishra-Gorur, 2019).

    Almost a century ago, two theories were stated to explain organotropic tumor metastasis. The 'anatomical mechanical theory' has bee proposed to account for cancer metastasis. It was theorized that the patterns of blood flow from the primary tumor can predict the first metastasized organs. On the other hand, the 'seed and soil' theory hypothesized that tumor cells migrate to tissues that support their growth. In other words, it was suggested that the site of metastasis depended on the affinity of the tumor for the microenvironment. A modern version of the seed and soil theory focuses on a 'homing mechanism', which suggests that tumor cells are drawn to specific organ sites because of complex signaling crosstalk between the tumor cells and the cells of the organ (Fidler, 2002). Consistent with this idea, recent studies in mammalian systems indicate that chemokines and their receptors are involved in organ-specific tumor metastasis (Mishra-Gorur, 2019).

    The strength of forward genetic screens and mosaic analysis in Drosophila has empowered investigators to utilize this model organism for identifying genes of cancer relevance, defining cancer signaling pathways and deciphering cancer biology. Previously a genetic screen was performed and a Drosophila model was developed for tumor metastasis (Pagliarini, 2003). Somatic cells expressing oncogenic Ras protein (RasV12) and simultaneously carrying a loss-of-function mutation in any of the cell polarity genes, including scribbled (scrib), lethal giant larvae (lgl) or discs large (dlg), develop into malignant tumors (known as RasV12/cell-polarity-defect tumors). This fly tumor model displays the main hallmarks of human metastatic cancers, including uncontrolled growth, basement membrane (BM) degradation, loss of E-cadherin, migration, invasion, and secondary-tumor formation at distant organs. Further studies have revealed that JNK (c-Jun N-terminal kinase) signaling is activated in the tumor cells and is required for tumor-cell migration and invasion. It was also learned that JNK signaling can be activated non-autonomously or under stress conditions and can propagate to collaborate with RasV12-expressing cells to induce tumorigenesis (Mishra-Gorur, 2019).

    This study consisted of a pathological and chronological examination of tumor progression and invasion in the fly tumor model. Tumor cells were found to metastasize to distal organs in a tissue-specific pattern. To identify genes responsible for organotropic metastasis, fly tumor cell lines were successfully established from the imaginal disc of a single larva, and a genome-wide RNA interference (RNAi) screen was performed. Toll-6, a Toll-receptor family member, was identified as a crucial gene for tumor cell migration. It was also shown that Toll-6 is required in tumor cells for organ-specific metastasis in vivo by inducing JNK signaling activation. Finally, the expression of the Spötzle (Spz) ligands in targeted organs serves as a cue for the guided migration and invasion. The Spz/Toll-6 system provides a novel molecular mechanism for organotropic metastasis (Mishra-Gorur, 2019).

    Metastasis is the leading cause of mortality in cancer patients. Befittingly, both the words 'cancer' (Latin: 'crab') and 'metastasis' (Greek: 'displacement') refer to cell movement: the crab-like invasion of cancer into healthy tissue and the migration of cancer cells to secondary sites. Since Paget's initial observation more than 100 years ago, pathologists have recognized that the movement of cancer cells is not random and that different types of cancer have different destinations or organ-specific metastasis. For example, colon carcinomas usually metastasize to liver and lung but rarely to bone, skin or brain and almost never to kidneys, intestine or muscle. In contrast, other tumors, such as breast carcinomas, frequently form metastases in most of these organs, while prostate cancer metastasis occurs most prominently in bone. A similar phenomenon occurs in the spread of malignant tumors in Drosophila. The RasV12/cell-polarity-defect tumors exhibit organ-specific metastasis. Tumor cells originating from the eye-antennal imaginal disc in the larvae metastasize to almost all organs (mouth hook, VNC, SG, leg discs, haltere disc, gut, FB) except the wing disc (Mishra-Gorur, 2019).

    In mouse cancer models and human patients, it is known that tumor cells are disseminated through vascular networks such as the blood and lymphatic vessels. The tracheal system in Drosophila is a tubular network for supplying oxygen, functioning as the equivalent of human lungs and blood vessels. Interestingly, in flies, it was also observed that tumor cells from primary tumors can metastasize to the trachea, suggesting that the trachea might function as an essential media to facilitate tumor metastasis. Consistent with this hypothesis, a recent study by Ross Cagan and Benjamin Levine showed that trachea-derived tumor cells can migrate significant distances (Levine, 2016). Additionally, RasV12/cell-polarity-defect tumors have been shown to express tracheal markers and migrate along the trachea (Grifoni, 2015). It will be interesting to further study the role of the trachea in organotropic metastasis, and possibly investigate whether a similar conserved mechanism exists in mammalian tumor cell migration (Mishra-Gorur, 2019).

    Targeted cell migration plays a key role in normal development. Studies of neural development have identified multiple receptors and their ligands that regulate guided neuronal migration (e.g. Robo/Slit, Trk receptors/neurotrophins). For organotropic metastasis, the 'seed and soil' theory states that metastases develop only when the 'seed' (tumor cells) and the 'soil' (target organs) are compatible. It is only recently that studies have begun to reveal some of the identities of the 'seed and soil' molecules that regulate the spread of tumor cells. Experiments with breast cancer cell lines showed that inhibition of the chemokine receptor CXCR4 by a neutralizing antibody abrogates their metastasis to the lung, which expresses the corresponding chemokine ligand, CXCL12. However, the CXCR4/CXCL12 interaction is unlikely involved in the metastasis of breast cancers to the liver The diverse types of cancers and their complex metastatic patterns indicate that other 'seed and soil' molecules must be involved in organ-specific metastasis (Mishra-Gorur, 2019).

    This study reports that Toll-6, a Toll-family neurotrophin receptor, plays an essential role in tumor cell metastasis in Drosophila. Toll-6 is expressed in the RasV12/cell-polarity-defect tumors, and downregulation of Toll-6 in the tumor cells blocks their migration and invasion. Interestingly, this study also showed that Toll-6-expressing tumor cells migrate towards and invade the organs that express Spz or Spz-related molecules. Furthermore, the Toll-6-expressing tumor cells do not metastasize to the wing disc, an organ with no detectable Spz or Spz-related gene expression in this system, strongly arguing that Spz or a Spz-related molecule could be serving as a cue for guiding Toll-6-expressing tumor cells. Interestingly, a recent study shows that the wing disc produces extremely low levels of Spz in a tightly regulated spatially restricted pattern with no resultant signaling events. Indeed, artificial overexpression of SpzACT in the wing disc converts it to a tissue receptive for the migration and invasion by Toll-6-expressing tumor cells. Together, these data indicate that Spz/Toll-6 serve as the 'seed and soil' molecules for organotropic metastasis in the fly. In support of this conclusion, clinical studies have revealed a correlation between increased expression levels of Toll-like receptors (TLRs) and malignancy of multiple cancers. TLRs have also been shown to affect malignancy by altering the tumor inflammatory microenvironment. Given that the Toll-family receptors are evolutionarily conserved, consisting of nine members in Drosophila and ten TLRs in mammals, this study raises the possibility that mammalian TLRs could play a similar role in mediating organotropic metastasis and cell migration (Mishra-Gorur, 2019).

    In Drosophila, Toll regulates dorsoventral patterning in embryos and anti-fungal defense in adults. Toll has also been found to have an inhibitory role in the formation of neuromuscular junctions and, recently, 18 wheeler, a Toll-like receptor protein, was found to play a role in border cell migration. Studies in both flies and mammals show that Toll-family receptors mediate NF-κB signaling activation. A study using biochemical inhibitors suggests that Toll molecules could affect random migration of neutrophils by activating ERKs. This study shows that Toll-6 activates JNK signaling, which is in accordance with the recent study by Foldi (2017) that shows the role of Toll-6 and JNK in cell death. However, JNK signaling is also a key regulator for cell migration during development, and previous work has shown that JNK signaling is activated in RasV12/cell-polarity-defect tumors and is essential for metastasis (Igaki, 2006). This study reports that, first, Toll-6 knockdown in RasV12/cell-polarity-defect tumors completely blocks metastasis by effectively reducing JNK signaling; second, the role of JNK activation by Toll-6 is highlighted in the in vitro assay, as Toll-6 knockdown reduced migration of the tumor cells even when discs were not placed, suggesting that JNK activation by Toll-6 might be important for general cell migratory behavior. Finally, ectopic expression of Toll-6ACT in the wing disc results in JNK activation. These data indicate that Toll-6 regulates metastasis by activating JNK signaling. Recently, Grindelwald (Grnd) has been identified as a novel tumor necrosis factor receptor (TNFR) mediating RasV12/scrib-/--induced tumor growth and invasion (Andersen, 2015). The current data suggest that Toll-6 could be providing a second signal for the activation of JNK. Indeed, epistasis data support the model that Toll-6 genetically interacts with the JNK pathway. It is possible that inputs from both Toll-6 and Grnd signals could result in or are required for a high level of JNK activation (Mishra-Gorur, 2019).

    In addition to Spz, there are five Spz-related genes in the Drosophila genome, which could serve as ligands for Toll-6. Toll-6 has been recently reported to function as a neurotrophin receptor in regulating motor neuron targeting and survival, and it also physically binds to Spz5. Consistently this study found that co-expression of Toll-6 and Spz5 synergistically promote collective cell invasion in the developing wing, phenocopying Toll-6 and SpzACT co-expression. Interestingly, although both SpzACT and Spz5 can activate Toll-6-mediated JNK signaling, it was found that, unlike Spz5, expression of SpzACT alone is not sufficient to activate JNK. Given the current results, it is inferred that Spz and Spz5 might display some redundancy. Indeed, a previous study by has demonstrated the redundancy of Spz2 (DNT1) and Spz5 (DNT2) in binding to Toll-6. Furthermore, a redundancy between Spz, Spz2 and Spz5 has also been demonstrated, suggesting that Spz proteins may function as promiscuous ligands in some circumstances (e.g., upon overexpression) and can bind multiple Toll receptors. Under overexpression conditions, Spz is thought to be capable of activating Toll-6 (albeit at lower levels than is Spz5). Therefore, this study finds that different Spz proteins might act redundantly to induce Toll-6-mediated JNK activation and cell migration. In conclusion, these genetic and biochemical data show that Toll-6 and Spz compose a new pair of guidance molecules for directing cell migration and that their interaction mediates organotropic metastasis by activating JNK signaling (Mishra-Gorur, 2019).

    Context-Dependent Tumorigenic Effect of Testis-Specific Mitochondrial Protein Tiny Tim 2 in Drosophila Somatic Epithelia

    A study was undertaken towards understanding the effect of ectopic expression of testis proteins in the soma in Drosophila. In the larval neuroepithelium, ectopic expression of the germline-specific component of the inner mitochondrial translocation complex tiny tim 2 (ttm2) brings about cell autonomous hyperplasia and extension of G2 phase. In the wing discs, cells expressing ectopic ttm2 upregulate Jun N-terminal kinase (JNK) signaling, present extended G2, become invasive, and elicit non-cell autonomous G2 extension and overgrowth of the wild-type neighboring tissue. Ectopic tomboy20, a germline-specific member of the outer mitochondrial translocation complex is also tumorigenic in wing discs. These results demonstrate the tumorigenic potential of unscheduled expression of these two testis proteins in the soma. They also show that a unique tumorigenic event may trigger different tumor growth pathways depending on the tissular context (Molnar, 2020).

    A Drosophila model of oral peptide therapeutics for adult Intestinal Stem Cell tumors

    Peptide therapeutics, unlike small molecule drugs, display crucial advantages of target-specificity and the ability to block large interacting interfaces such as those of transcription factors. The transcription co-factor of the Hippo pathway, YAP/Yki, has been implicated in many cancers, and is dependent on its interaction with the DNA-binding TEAD/Sd proteins via a large Ω-loop. In addition, the mammalian Vestigial Like (VGLL) protein, specifically its TONDU domain, competitively inhibits YAP-TEAD interaction, resulting in arrest of tumor growth. This study shows that either overexpression of the TONDU peptide or its oral uptake leads to suppression of Yorkie (Yki)-driven intestinal stem cell (ISC) tumors in the adult Drosophila midgut. In addition, comparative proteomic analyses of peptide-treated and untreated tumors, together with ChIP analysis, reveal that integrin pathway members are part of the Yki-oncogenic network. Collectively, these findings establish Drosophila as a reliable in vivo platform to screen for cancer oral therapeutic peptides and reveal a tumor suppressive role for integrins in Yki-driven tumors (Bajpai, 2020).

    Systemic muscle wasting and coordinated tumour response drive tumourigenesis

    Cancer cells demand excess nutrients to support their proliferation, but how tumours exploit extracellular amino acids during systemic metabolic perturbations remain incompletely understood. This study used a Drosophila model of high-sugar diet (HSD)-enhanced tumourigenesis to uncover a systemic host-tumour metabolic circuit that supports tumour growth. Coordinate induction is demonstrated of systemic muscle wasting with tumour-autonomous Yorkie-mediated SLC36-family amino acid transporter expression as a proline-scavenging programme to drive tumourigenesis. Indole-3-propionic acid was identified as an optimal amino acid derivative to rationally target the proline-dependency of tumour growth. Insights from this whole-animal Drosophila model provide a powerful approach towards the identification and therapeutic exploitation of the amino acid vulnerabilities of tumourigenesis in the context of a perturbed systemic metabolic network (Newton, 2020).

    Cancer cells require a constant supply of metabolic intermediates to support their proliferation. To meet the biosynthetic demands associated with tumourigenesis, cancer cells actively acquire nutrients from the extracellular space. Cancer is a systemic disease that associates with a range of host metabolic abnormalities such as obesity, insulin resistance and cancer-associated cachexia; each of which alters the host systemic nutritional environment. These changes in both nutrient composition and availability may have profound effects on cancer development and progression. However, how cancer cells sense and respond to nutritional changes in the context of organismal metabolic alterations remains an underexplored area in cancer biology (Newton, 2020).

    Cancer-associated cachexia is a systemic metabolic syndrome of weight loss associated with progressive skeletal muscle wasting. The multifactorial and heterogeneous condition of cachexia involves a complex multi-organ interplay, which has impeded its comprehensive understanding at the molecular level. A series of recent studies using Drosophila melanogaster have shown that tumour-derived factors modulate host metabolism. In addition, tumour-derived factors promote the release of nutrients from the tumour microenvironment to promote tumour growth. This study has leveraged a Drosophila model of high-sugar diet (HSD)-enhanced tumourigenesis and demonstrates that HSD-enhanced tumours induce SLC36-family transporter expression as a coordinated mechanism to exploit exogenous proline for tumourigenesis during systemic muscle wasting. Furthermore, these mechanistic insights were used to rationally target the proline dependency of tumours as an approach to inhibit tumour growth (Newton, 2020).

    Muscle wasting is observed in chronic muscle wasting diseases due to cancer (cachexia), ageing/senescence (sarcopenia), myopathies and other metabolic diseases, as well as in acute conditions due to burns and sepsis. This study demonstrates that the combination of diet-induced obesity and tumour growth induces systemic muscle wasting, associated with functional locomotion defect. This muscle phenotype was not due to the inability to properly form muscle during development; the muscles are wholly formed at the early stage of larval development, and waste progressively at the later larval stage, as the tumours develop. In addition, it was demonstrated that the muscle phenotype observed in this model is unrelated to a developmentally related degeneration process (Newton, 2020).

    This study reveals a systemic amino acid-utilising circuit whereby HSD-enhanced tumours induce muscle wasting as a systemic metabolic network to drive tumourigenesis. A consequence of muscle wasting in ras1G12V;csk-/- animals raised on HSD was increased release of proline into the circulation. Plasma amino acid profiling of patients with sarcopenia - a condition of muscle wasting associated with aging—showed elevated plasma proline levels, indicating that elevated free circulating proline is a common feature of muscle wasting. This study identified a proline vulnerability of HSD-enhanced tumours; SLC36-family amino acid transporter Path is required for tumour growth and exogenous proline promotes tumourigenesis through Path. This study highlights two layers of coordination in tumour metabolic response: (1) at the whole organism level, by promoting muscle wasting and systemic amino acid availability, and (2) at the tumour-autonomous level, by altering amino acid transporter repertoire (Newton, 2020).

    Path and CG1139 have different transport characteristics for proline, with Path having a lower transport capacity compared to CG1139. Despite this, labelled proline uptake experiments indicated uptake of extracellular proline in Ras/Src/Path-tumours but not in Ras/Src/CG1139-tumours, suggesting that CG1139 and Path may exhibit different activities in the context of an oncogenic background. Consistent with a previous report (Goberdhan, 2005), the current data support the signalling role of Path through activation of the Tor-S6K pathway. Proline metabolism has been demonstrated to support cancer clonogenicity and metastasis. Furthermore, proline promotes cancer cell survival under nutrient-limited or hypoxic microenvironments. The current data extend these studies to reveal a tumour-promoting role of proline in response to systemic host metabolic changes (Newton, 2020).

    Indole-3-propionic acid (IPA) specifically targets the proline dependency of tumour growth in Drosophila. Furthermore, this study demonstrates a functional effect of IPA on human cells. A recent study demonstrated that proline uptake in Ras-driven tumour cells is much higher in spheroids in three-dimensional cultures—which better mimic conditions in vivo—compared to monolayers in two-dimensional cultures, suggestive of a potentially stronger effect in tumours that are dependent on proline for growth in vivo. Proline is a limiting amino acid for protein synthesis in kidney cancers. Therefore, reducing proline uptake with SLC36-inhibitors -- as exemplified here by use of IPA -- may warrant consideration as a therapeutic strategy to break the nutritional circuit between systemic muscle wasting and tumour growth in proline vulnerable cancers (Newton, 2020).

    Role for phagocytosis in the prevention of neoplastic transformation in Drosophila

    Immunity is considered to be involved in the prevention of cancer. Although both humoral and cellular immune reactions may participate, underlying mechanisms have yet to be clarified. The present study was conducted to clarify this issue using a Drosophila model, in which neoplastic transformation was induced through the simultaneous inhibition of cell-cycle checkpoints and apoptosis. First, the location was determined of hemocytes, blood cells of Drosophila playing a role of immune cells, in neoplasia-induced and normal larvae, but there was no significant difference between the two groups. When gene expression pattern in larval hemocytes was determined, the expression of immunity-related genes including those necessary for phagocytosis was reduced in the neoplasia model. Then the involvement of phagocytosis was determined in the prevention of neoplasia, examining animals where the expression of engulfment receptors (Draper and integrin αPS3/βν) instead of apoptosis was retarded. It was found that the inhibition of engulfment receptor expression augmented the occurrence of neoplasia induced by a defect in cell-cycle checkpoints. This suggested a role for phagocytosis in the prevention of neoplastic transformation in Drosophila (Zhang, 2020).

    Coopted temporal patterning governs cellular hierarchy, heterogeneity and metabolism in Drosophila neuroblast tumors

    It is still unclear what drives progression of childhood tumors. During Drosophila larval development, asymmetrically-dividing neural stem cells, called neuroblasts, progress through an intrinsic temporal patterning program that ensures cessation of divisions before adulthood. Previous work has shown that temporal patterning also delineates an early developmental window during which neuroblasts are susceptible to tumor initiation. Using single-cell transcriptomics, clonal analysis and numerical modeling, this study now identifies a network of twenty larval temporal patterning genes that are redeployed within neuroblast tumors to trigger a robust hierarchical division scheme that perpetuates growth while inducing predictable cell heterogeneity. Along the hierarchy, temporal patterning genes define a differentiation trajectory that regulates glucose metabolism genes to determine the proliferative properties of tumor cells. Thus, partial redeployment of the temporal patterning program encoded in the cell of origin may govern the hierarchy, heterogeneity and growth properties of neural tumors with a developmental origin (Genovese, 2019).

    Central nervous system (CNS) tumors are rare and constitute less than 2% of all cancers in adults. In contrast, they represent more than 25% of cancer cases in children (including medulloblastoma, retinoblastoma, rhabdoid tumors (AT/RT), gliomas etc), suggesting that the developing CNS is particularly sensitive to malignant transformation. Moreover, unlike most adult tumors, pediatric tumors are often genetically stable and their initiation and progression do not necessarily require the accumulation of mutations in multiple genes. For example, the biallelic inactivation of a single gene is sometimes sufficient to trigger malignant growth as illustrated by mutations in the RB1 and SMARCB1 genes in retinoblastoma and rhabdoid tumors respectively. Recent studies suggest that CNS pediatric tumors such as medulloblastomas recapitulate the fetal transcription program that was active in the cell of origin. However, it remains unclear how the invalidation of single genes during fetal stages can disrupt on-going developmental programs to trigger malignant growth, and whether these fetal/developmental programs influence the heterogeneity, composition, and proliferative properties of cells composing CNS tumors (Genovese, 2019).

    Faced with the complexity of brain development and neural tumors in mammals, simple animal models can represent a powerful alternative to investigate basic and evolutionary conserved principles. The development of the CNS is undoubtedly best understood in Drosophila. The Drosophila CNS arises from a small pool of asymmetrically-dividing neural stem cells (NSCs), called neuroblasts (NBs). NBs possess a limited self-renewing potential. They divide all along development (embryonic and larval stages) to self-renew while generating daughter cells named Ganglion Mother Cells (GMCs). GMCs then usually divide once to produce two post-mitotic neurons or glia. NBs are the fastest cycling cells during development, able to divide every hour during larval stages when most of the neurons are produced. However, all NBs terminate during metamorphosis and are absent in adults. Two antagonistic RNA-binding proteins, IGF-II mRNA-binding protein (Imp) and Syncrip (Syp) are essential to first promote and then conclude this formidable period of activity. During early larval development (L1/L2), NBs express Imp that promotes NB self-renewal. Around late L2/early L3, NBs silence Imp to express Syp that remains expressed until NB decommissioning during metamorphosis. This Imp-to-Syp transition is essential to render NBs competent to respond to subsequent pupal pulses of the steroid hormone ecdysone and initiate a last differentiative division. Failure to trigger the transition results in NBs permanently dividing in adults. The Imp-to-Syp transition appears to be mainly regulated by a NB intrinsic timing mechanism driven by the sequential expression of transcription factors. This series of factors, also known as temporal transcription factors, has been first identified for its ability to specify different neuronal fates produced by NBs as they divide. In addition, temporal transcription factors also schedule the Imp-to-Syp transition to ensure that NBs will not continue cycling in adults. Recent transcriptomic analyses indicate that other genes are dynamically transcribed in NBs throughout larval stages, although their function and epistatic relationship with temporal transcription factors and the Imp/Syp module are unclear. All together, these studies highlight a complex, but still relatively unexplored, temporal patterning system in larval NBs (Genovese, 2019).

    Perturbation of the asymmetric division process during early development can lead to NB exponential amplification. In such conditions, the NB-intrinsic temporal program limiting self-renewal appears to become inoperant, and uncontrolled NB amplification is observed. Serial transplantations of asymmetric division-defective NBs have revealed an ability to proliferate for months, if not years, demonstrating tumorigenic characteristics. Perturbation of asymmetric divisions can be induced by the inactivation of the transcription factor Prospero (Pros) in type I NB lineages (most lineages in the ventral nerve cord (VNC) and central brain (CB)). During development, Pros is strongly expressed in GMCs where it accumulates to induce cell cycle-exit and neuronal or glial differentiation. GMCs that lack pros fail to differentiate and revert to a NB-like state. This triggers rapid NB amplification at the expense of neuron production. Previous work has shown that inactivation of pros in NBs, and their subsequent GMCs, before mid-L3 (L3 being the last larval stage) leads to aggressive NB tumors that persist growing in adults. In contrast, inactivation of pros after mid-L3 leads to transient NB amplification and most supernumerary NB properly differentiate during metamorphosis, leading to an absence of growing tumors in adults. Interestingly, propagation of NB tumor growth beyond normal developmental stages is caused by the aberrant maintenance of Imp and the transcription factor Chinmo from early-born GMCs, the latter representing the cells of origin of such aggressive tumors (Narbonne-Reveau, 2016). Chinmo and Imp positively cross-regulate and inactivation of either in NB tumors stops tumorigenic growth. Because pros-/- NB tumors can only be induced during an early window of development, and are caused by the biallelic inactivation of a single gene, they represent an exciting and simple model to investigate the basic mechanisms driving the growth of tumors with an early developmental origin, such as in the case of pediatric CNS cancers (Genovese, 2019).

    NB tumors can also be induced from type II NBs (a small subset of NBs in the central brain) or from neurons upon inactivation of the NHL-domain family protein Brat or Nerfin-1 respectively. In both cases, tumor growth appears to rely on the aberrant expression of the Chinmo/Imp module arguing for a general tumor-driving mechanism in the developing Drosophila CNS (Narbonne-Reveau, 2016). Interestingly, in the different types of NB tumors, Chinmo and Imp are only expressed in a subpopulation of cells, demonstrating heterogeneity in the population of tumor NBs (tNBs). However, the full repertoire of cells composing the tumor, the rules governing the cellular heterogeneity and the mechanisms determining the proliferative potential of each cell type remain to be investigated (Genovese, 2019).

    This study used single-cell RNA-seq, clonal analysis and numerical modeling to investigate these questions. A subset of genes involved in the temporal patterning of larval NBs were identified that are redeployed in tumors to generate a differentiation trajectory responsible for creating tumor cell heterogeneity. This cellular heterogeneity results in NBs with different types of metabolism and different proliferative properties. This study also deciphered a robust hierarchical scheme that drives reproducible heterogeneity through the dysregulated but fine-tuned transition between the two RNA-binding proteins Imp and Syp. This work thus identifies a core larval NB temporal patterning program, the disruption of which not only causes unlimited growth but has an overarching role in governing the cellular hierarchy, heterogeneity and metabolism of NB tumors (Genovese, 2019).

    This study demonstrates that temporal patterning, not only determines which cells are susceptible to cancer transformation during development (Narbonne-Reveau, 2016), but also has an overarching role in governing different aspects of CNS tumor organization such as hierarchy, heterogeneity and the proliferative properties of the different types of cells via the regulation of their metabolism (Genovese, 2019).

    Given the recent discovery that temporal patterning is conserved in the developing mammalian brain (Telley, 2019), this study could shed light on an ancestral mechanism that governs the progression of CNS tumors with developmental origins (Genovese, 2019).

    The rules governing the initiation and progression of CNS pediatric tumors that often exhibit stable genomes are still unclear. Previousl work has demonstrated that temporal patterning in Drosophila larval NBs delineates a window of time during which the Chinmo/Imp oncogenic module is expressed and makes early larval NBs prone to malignant transformation (Narbonne-Reveau, 2016). This study finds that after tumor initiation, temporal patterning is partly recapitulated in tNBs where it generates differentiation trajectories to constrain tumor composition and growth. This is illustrated by the presence of about 20 genes (Imp, chinmo, Lin-28, E23, Oatp74D, Gapdh2, Sip1/CG10939, plum/CG6490, SP1173, Chd64, CG10512, CG44325, CG5953, Syp, E93, lncRNA:noe, CG15646 and stg), previously identified to be temporally regulated in some larval NBs, that are differentially regulated along the pseudotime/differentiation trajectory reconstructed from single-cell RNA-seq analysis of tNBs, and/or differentially expressed in Imp+ vs Syp+ tNBs. Thus, this study identified what appears to be a subset of a core temporal patterning program encoded in central brain and ventral nerve cord NBs that becomes deregulated upon asymmetric-division defects during early development (Genovese, 2019).

    Notably, the larval temporal transcription factor Cas and Svp, known to schedule the Imp-to-Syp transition during development are not enriched in Imp+ tNBs suggesting that they do not play a role in regulating the Imp-to-Syp transition along the trajectory in tumors. Interestingly, while Syp is transcriptionally regulated in larval NBs, it seems rather post-transcriptionally regulated in tNBs as Syp RNAs are present throughout all clusters. This suggests that different mechanisms may be operating in tumors than during development to regulate the Imp-to-Syp transition (Genovese, 2019).

    This study observed that the proportions of Imp+ and Syp+ tNBs systematically reach an equilibrium over a few days with a 20/80 (+/-10) ratio in poxn > prosRNAi tumors. This suggests that the regulation of the Imp-to-Syp transition in tumors is not random and the predictability of the final proportions possibly implies robust underlying constraints. By investigating the population dynamics of Imp+ and Syp+ tNBs in prosRNAi tumors, this study has deciphered a finely tuned hierarchical division scheme that appears to constrain the growth and cellular heterogeneity of the tumor. Imp+ tNBs is shown in the tumorigenic context favor a symmetric self-renewing mode of divisions (in more than 60% of divisions) while unlikely to exit the cell-cycle. This allows the perpetuation of a small subset of Imp+ tNBs that are endowed with a seemingly unlimited self-renewing potential by the Imp/Chinmo module. Imp+ tNBs can also make symmetric and asymmetric divisions that generate Syp+ tNBs, leading to the production of a population of Syp+E93+ tNBs that accumulates through limited self-renewal, and have a high propensity for exiting the cell-cycle. Moreover, this study could shows that Syp+E93+ tNBs are unable to generate Imp+ tNBs, demonstrating a rigid cellular hierarchy reminiscent of development. In addition, in this context, Syp acts as a tumor suppressor by limiting tNB proliferation while Imp acts as an oncogene by promoting tNB proliferation and propagation of tumor growth. Together, these data argue for a scenario where cooption of the Imp-to-Syp transition is responsible for installing a hierarchical mode of tumor growth with Imp+ tNBs propagating unlimited growth in a CSC-like manner, while Syp+E93+ tNBs acts as transient amplifying progenitors with limited self-renewing abilities. Although the Imp/Syp RNA-binding proteins have an essential and antagonistic role in governing the proliferative properties of tumor cells, the function of the other redeployed temporal patterning genes is unknown (except for chinmo, downstream to Imp and Syp, that is essential for tumor growth). As many are linked with the Imp+ tNB state, it will be important in the future to decipher how they contribute to establish or maintain the CSC-like identity (Genovese, 2019).

    The division parameters defined by clonal analysis and modeling approach could capture both the hierarchical aspect of tumor growth as well as the global population dynamics: from an initial homogenous pool of larval Imp+ tNBs to the stable heterogeneity observed during adulthood. It could also resolve the paradoxical observation that Chinmo+Imp+ tNBs end up in minority despite exhibiting a higher average mitotic rate. Although, like all models, it is not expected that this model would perfectly recapitulate all the parameters regulating tumor growth and heterogeneity (for example, this study has neglected apoptosis and neuronal differentiation that occur at low levels), it is thought that this model provides a reasonable and useful ground on which further studies can be performed for a more detailed understanding. On these lines, while the division pattern this study has described with a numerical model provides estimates of division probabilities in poxn > prosRNAi tumors, it says nothing as to how these probabilities are biologically set within the tumor. A possible scenario is that cell fate determination upon division relies on signals received by immediate neighboring tumor cells, resulting in effective probabilities at the scale of the whole tumor. Such a micro-environment dependent regulation of the Imp-to-Syp transition in tumors would strongly contrast with the cell-intrinsic regulation of the Imp-to-Syp transition that systematically occurs in NBs around early L3. Future studies will aim at deciphering the mechanisms that interfere with the developmental progression of the temporal patterning, upon asymmetric-division defects, to favor the self-renewing mode of divisions undergone by the Chinmo+Imp+ tNBs, allowing perpetuation of a population of CSC-like cells (Genovese, 2019).

    Noteworthy, prosRNAi and snr1/dSmarcb1RNAi tumors exhibit different but reproducible ratios of Imp+ and Syp+ tNBs. This suggests the existence of tumor-specific mechanisms that fine-tune the Imp-to-Syp transition. Such mechanisms may be related to the tumor cell of origin, or to the genetic insult that initiated NB amplification. Further analysis will help identifying tumor-intrinsic signals regulating the balance between Chinmo+Imp+ tNBs and Syp+E93+ tNBs in various types of NB tumors (Genovese, 2019).

    Until recently, the existence of temporal patterning in mammalian neural progenitors remained uncertain. Elegant single-cell transcriptomic studies of embryonic cortical and retinal progenitors in mice have now revealed that they transit through different transcriptional states that are transmitted to their progeny to generate neuronal diversity, similar to temporal patterning in Drosophila (Clark, 2019; Telley, 2019). However, it remains unknown whether temporal patterning determines the cell of origin and governs the growth of CNS tumors in children. Along these lines, the finding that the transcriptional programs operating in cerebellar progenitors during fetal development are recapitulated in medulloblastomas is promising (Vladoiu, 2019). By uncovering the overarching role of temporal patterning in governing tumor susceptibility during CNS development and in constraining tumor properties during cancer progression in Drosophila, this work thus possibly provides a new conceptual framework to better understand CNS tumors in children (Genovese, 2019).

    Because of the difficulty to investigate metabolism at the single-cell level, it has been difficult to determine how heterogeneous is the metabolic activity of cells in tumors, and how it is controlled. Using a combination of single-cell and bulk RNA-seq approaches, this study has found that progression of temporal patterning provides a tumor-intrinsic mechanism that generates heterogeneity in the proliferative abilities of tumor cells through the progressive silencing of glucose and glutamine metabolism genes (Genovese, 2019).

    Consequently, Chinmo+Imp+ tNBs, that lie at the top of the hierarchy, highly express glycolytic and respiratory/OXPHOS genes, as well as Gdh, that are down-regulated by the Imp-to-Syp transition. This default high expression of both glutamine and glucose metabolism genes in CSC-like Chinmo+Imp+ tNBs likely favors sustained self-renewal, but could also confer plasticity and a way to adapt cellular metabolism to different environmental conditions as frequently observed in CSCs (e.g., glutamine can compensate for glucose shortage) (Sancho, 2016) (Genovese, 2019).

    This study showed that Syp+E93+ tNBs exhibit a reduced size, and that knock-down of glycolytic (Gapdh1 or Pglym78) or respiratory/OXPHOS genes (Cyt-c-p or Cyt-C1) prevented propagation of tumor growth in adults. Thus, reduction of biosynthesis and energy production through down-regulation of glucose and glutamine metabolism genes after the Imp-to-Syp transition could progressively exhaust Syp+E93+ tNB growth and self-renewing ability, ultimately leading to cell-cycle exit (Genovese, 2019).

    With the demonstration that temporal patterning regulates glycolytic, TCA cycle and OXPHOS genes in NB tumors, this work provides a tumor-intrinsic mechanism that creates metabolic heterogeneity to control the proliferative potential of the various tumor cells. It was also observed that Syp+E93+ tNBs associated with lowest levels of metabolic and cell-cycle genes also upregulate genes of the E(spl) genes. Interestingly, expression of Hes genes (orthologs of Enhancer of split genes) in vertebrate neural stem cells is associated with the maintenance of a quiescent state in adults. Thus, E(spl) genes may promote the quiescent tNB state identified with the clonal and numerical analysis while preventing their differentiation in neurons (Genovese, 2019).

    Down-regulation of the mRNA levels of metabolic genes after the Imp-to-Syp transition could be due to the silencing of a transcriptional activator or to an increased mRNA degradation. On one hand, Chinmo is a likely candidate for the first scenario, as its inactivation reduces growth in NBs (Narbonne-Reveau, 2016) and this study showed that it is a direct target of both Imp and Syp. On the other hand, the second scenario is consistent with Imp orthologs in human being able to promote OXPHOS and proliferation in glioma cells, through the post-transcriptional regulation of mitochondrial respiratory chain complex subunits (Genovese, 2019).

    This study has also identified a small population of tNBs expressing various stress or growth arrest factors. One of these factors, Xrp1, is a transcriptional target of p53 in the response to irradiation. Xrp1 expression has also recently been linked to defects in translation rates, together with the expression of Irbp18 and GstE6. Thus, these factors may label a subset of tNBs undergoing DNA or translational stress. The reason and consequences of such cellular stresses in tumor progression need to be further investigated (Genovese, 2019).

    Transcriptomic analyses have revealed strong similarities in the differentiation trajectories of tNBs in tumors and of NBs in larvae. Yet, it is surprising that the down-regulation of glutamine and glucose metabolism genes has not been detected in NBs during larval development, after the Imp-to-Syp transition (Ren, 2017). It is possible that the glial niche surrounding NBs, that is known to influence NB growth properties during larval stages, somehow sustains high levels of glucose metabolism genes in late Syp+E93+ NBs. Given that this glial niche is absent in tumors, Syp+E93+ tNBs may not be able to sustain the high expression of metabolic genes imposed by the Imp/Chinmo module, leading to progressive cell-cycle exit (Genovese, 2019).

    Chinmo and Imp are reminiscent to oncofetal genes in mammals, in that their expression decrease rapidly as development progresses while they are mis-expressed in tumors. Along these lines, the three IMP orthologs in humans (also called IGF2BP1-3) are also known as oncofetal genes. They emerge as important regulators of cell proliferation and metabolism in many types of cancers including pediatric neural cancers. Along evolution, the ancestral Syncrip gene has been subjected to several rounds of duplication and has diverged into five paralogs in mammals, some of them emerging as tumor suppressors with an important role in tumor progression (Genovese, 2019).

    Thus, the respective oncogenic and tumor suppressor roles of IMP and SYNCRIP gene families appear to have been conserved in humans and they may not be restricted to tumors of neural origin. This study therefore raises the exciting possibility that these two families of RNA-binding proteins form a master module at the top of the self-renewal/differentiation cascades, that regulates CSC populations and hierarchy in a spectrum of human cancers (Genovese, 2019).

    The transcription factor spalt and human homologue SALL4 induce cell invasion via the dMyc-JNK pathway in Drosophila

    Cancer cell metastasis is a leading cause of mortality in cancer patients. Therefore, revealing the molecular mechanism of cancer cell invasion is of great significance for the treatment of cancer. In human patients, the hyperactivity of transcription factor Spalt-like 4 (SALL4) is sufficient to induce malignant tumorigenesis and metastasis. This study found that when ectopically expressing the Drosophila homologue spalt (sal) or human SALL4 in Drosophila, epithelial cells delaminated basally with penetration of the basal lamina and degradation of the extracellular matrix, which are essential properties of cell invasion. Further assay found that sal/SALL4 promoted cell invasion via dMyc-JNK signaling. Inhibition of the c-Jun N-terminal kinase (JNK) signaling pathway through suppressing matrix metalloprotease 1 or basket can achieve suppression of cell invasion. Moreover, expression of dMyc, a suppressor of JNK signaling, dramatically blocked cell invasion induced by sal/SALL4 in the wing disc. These findings reveal a conserved role of sal/SALL4 in invasive cell movement and link the crucial mediator of tumor invasion, the JNK pathway, to SALL4-mediated cancer progression (Sun, 2020).

    Notch mediates inter-tissue communication to promote tumorigenesis

    Disease progression in many tumor types involves the interaction of genetically abnormal cancer cells with normal stromal cells. Neoplastic transformation in a Drosophila genetic model of Epidermal growth factor receptor (EGFR)-driven tumorigenesis similarly relies on the interaction between epithelial and mesenchymal cells, providing a simple system to investigate mechanisms used for the cross-talk. Using the Drosophila model, this study shows that the transformed epithelium hijacks the mesenchymal cells through Notch signaling, which prevents their differentiation and promotes proliferation. A key downstream target in the mesenchyme is Zfh1/ZEB. When Notch or zfh1 are depleted in the mesenchymal cells, tumor growth is compromised. The ligand Delta is highly upregulated in the epithelial cells where it is found on long cellular processes. By using a live transcription assay in cultured cells and by depleting actin-rich processes in the tumor epithelium, this study provides evidence that signaling can be mediated by cytonemes from Delta-expressing cells. It is thus proposed that high Notch activity in the unmodified mesenchymal cells is driven by ligands produced by the cancerous epithelial. This long-range Notch signaling integrates the two tissues to promote tumorigenesis, by co-opting a normal regulatory mechanism that prevents the mesenchymal cells from differentiating (Boukhatmi, 2020).

    Normal tissue mesenchymal cells are thought to have important roles in promoting the growth and metastasis of many tumors. To do so, they must be educated by the aberrant cancerous cells to acquire the properties needed to sustain tumorigenesis. Using a Drosophila model of EGFR/Ras-driven tumorigenesis, this study demonstrates that Notch activity in the unmodified mesenchymal cells is essential for tumor growth. Downregulating Notch specifically in mesenchymal cells reduced their proliferation rates, promoted their differentiation, and significantly compromised the size of tumors that developed. Strikingly, the activation of Notch in these supporting cells appears to rely on direct communication from the cancerous epithelial cells, illustrating that this pathway can operate in long-range signaling between tissue layers (Boukhatmi, 2020).

    The conclusion that Notch receptors in the mesenchymal cells are activated from ligands presented by nearby epithelial cells is unexpected because most examples of Notch signaling occur between cells within an epithelial cell layer. The fact that the ligands are transmembrane proteins means that direct cell-cell contacts are required to elicit signaling and that signaling usually occurs between neighboring cells. More recently, examples have emerged where signaling occurs across longer distances that appear to involve contacts mediated by cell protusions, such as filopodia or cytonemes. Evidence indicates that a similar mechanism operates in the tumors. Delta is produced in the epithelial cells and can be detected in fine processes that extend through the nearby mesenchymal cells, which is consistent with a recent report describing cytonemes in these EGFR-psqRNAi tumors. In a heterologous system, it was found there was robust activation of a Notch target gene rapidly after ligand-expressing cells made contact through cell processes. Likewise, ectopic patches of Delta in the disc epithelium led to the expression of the Notch-regulated m6-GFP in the underlying mesenchyme. Thus, it is proposed that the widespread upregulation of Delta in the epithelial compartment of the tumorous wing discs, in turn, activates the Notch pathway in the neighboring mesenchymal cells by long cellular processes. As a consequence, the mesenchymal cells become coordinated with the cancer epithelial cells and are maintained in an undifferentiated state (Boukhatmi, 2020).

    Although the data demonstrate that Delta-Notch-mediated inter-tissue signaling is important for sustaining tumor growth, it is evident that other signals are also required. First, it was previously shown that Dpp from the cancerous epithelium is essential for these tumors to grow. Because the Dpp pathway was still activated in the mesenchyme when Notch was depleted, it is proposed that Dpp and Notch operate in parallel. This may explain why apicobasal polarity was not fully restored when Notch activity was impaired and highlights the likelihood that several different pathways are coopted to drive tumorigenesis. Second, the fact that tumorigenesis is rescued by perturbing Notch or Dpp signaling in the mesenchyme argues that there must be a reciprocal signal to the epithelium. Notably, the relative growth of the two populations appears highly co-ordinated in the tumors, unlike the wild type where the epithelial growth predominates. A plausible model is that combined inputs from Notch and Dpp are required to produce reciprocal signal(s), and it will be interesting to discover whether the reciprocal signaling also operates through cytonemes, given that the mesenchymal cells emit processes (Boukhatmi, 2020).

    One of the key effectors of Notch activity in the tumor mesenchyme is Zfh1/ZEB, which is important for maintaining the muscle progenitors in normal conditions. In a similar manner, its expression is kept high in the tumor mesenchyme, due to Notch activity, where it helps prevent their differentiation. Downregulating zfh1 in mesenchymal cells induces their premature differentiation and prevents tumor growth. The role of Zfh1/ ZEB in promoting progenitors and stem cell proliferation appears to be widespread. Furthermore, ZEB1 is upregulated in many cancers, where it can cause the expansion of cancer stem cells and frequently drives epithelial-to-mesechymal transition to promote metastasis. Whether its activation in these conditions also involves Notch activation and inter-tissue signaling remains to be determined (Boukhatmi, 2020).

    Genome Wide Screen for Context-Dependent Tumor Suppressors Identified Using in Vivo Models for Neoplasia in Drosophila

    This study reports on large-scale RNAi-based screens to identify potential tumor suppressor genes that interact with known cancer-drivers: the Epidermal Growth Factor Receptor and the Hippo pathway transcriptional cofactor Yorkie. These screens were designed to identify genes whose depletion drove tissue expressing EGFR or Yki from a state of benign overgrowth into neoplastic transformation in vivo. An independent screen aimed to identify genes whose depletion suppressed formation of neoplastic tumors in an existing EGFR-dependent neoplasia model. Many of the positives identified here are known to be functional in growth control pathways. A number of novel connections to Yki and EGFR driven tissue growth were identified, mostly unique to one of the two. Thus, resources provided in this study would be useful to all researchers who study negative regulators of growth during development and cancer in the context of activated EGFR and/or Yki and positive regulators of growth in the context of activated EGFR. Resources reported here are available freely for anyone to use (Groth, 2020).

    A Genetic Analysis of Tumor Progression in Drosophila Identifies the Cohesin Complex as a Suppressor of Individual and Collective Cell Invasion

    Metastasis is the leading cause of death for patients with cancer. Consequently it is imperative to improve understanding of the molecular mechanisms that underlie progression of tumor growth toward malignancy. Advances in genome characterization technologies have been very successful in identifying commonly mutated or misregulated genes in a variety of human cancers. However, the difficulty in evaluating whether these candidates drive tumor progression remains a major challenge. Using the genetic amenability of Drosophila melanogaster, this study generated tumors with specific genotypes in the living animal and carried out a detailed systematic loss-of-function analysis to identify conserved genes that enhance or suppress epithelial tumor progression. This enabled the discovery of functional cooperative regulators of invasion and the establishment of a network of conserved invasion suppressors. This includes constituents of the cohesin complex (see Rad21). whose loss of function either promotes individual or collective cell invasion, depending on the severity of effect on cohesin complex function (Canales Coutino, 2020).

    NatB regulates Rb mutant cell death and tumor growth by modulating EGFR/MAPK signaling through the N-end rule pathways

    Inactivation of the Rb tumor suppressor causes context-dependent increases in cell proliferation or cell death. In a genetic screen for factors that promoted Rb mutant cell death in Drosophila, this study identified Psid, a regulatory subunit of N-terminal acetyltransferase B (NatB). NatB subunits were required for elevated EGFR/MAPK signaling and Rb mutant cell survival. NatB regulates the posttranscriptional levels of the highly conserved pathway components Grb2/Drk, MAPK, and PP2AC but not that of the less conserved Sprouty. Interestingly, NatB increased the levels of positive pathway components Grb2/Drk and MAPK while decreased the levels of negative pathway component PP2AC, which were mediated by the distinct N-end rule branch E3 ubiquitin ligases Ubr4 and Cnot4, respectively. These results suggest a novel mechanism by which NatB and N-end rule pathways modulate EGFR/MAPK signaling by inversely regulating the levels of multiple conserved positive and negative pathway components. As inactivation of Psid blocked EGFR signaling-dependent tumor growth, this study raises the possibility that NatB is potentially a novel therapeutic target for cancers dependent on deregulated EGFR/Ras signaling (Sheng, 2020).

    A Forward Genetic Approach to Mapping a P-Element Second Site Mutation Identifies DCP2 as a Novel Tumour Suppressor in Drosophila melanogaster

    The use of transposons to create mutations has been the cornerstone of Drosophila genetics in the past few decades. Second-site mutations caused by transpositions are often devoid of transposons and thereby affect subsequent analyses. In a P-element mutagenesis screen, a second site mutation was identified on chromosome 3, wherein the homozygous mutants exhibit classic hallmarks of tumour suppressor mutants, including brain tumour and lethality; hence the mutant line was initially named as lethal (3) tumorous brain [l(3)tb]. Classical genetic approaches relying on meiotic recombination and subsequent complementation with chromosomal deletions and gene mutations mapped the mutation to CG6169, the mRNA decapping protein 2 (DCP2), on the left arm of the third chromosome (3L). Thus the mutation was renamed as DCP2(l(3)tb) Fine mapping of the mutation further identified the presence of a Gypsy--LTR like sequence in the 5'UTR coding region of DCP2, along with the expansion of the adjacent upstream intergenic AT-rich sequence. The mutant phenotypes are rescued by the introduction of a functional copy of DCP2 in the mutant background, thereby establishing the causal role of the mutation and providing a genetic validation of the allelism. With the increasing repertoire of genes being associated with tumour biology, this is the first instance of mRNA decapping protein being implicated in Drosophila tumourigenesis. These findings, therefore, imply a plausible role for the mRNA degradation pathway in tumorigenesis and identify DCP2 as a potential candidate for future explorations of cell cycle regulatory mechanisms (Mishra, 2020).

    Transcriptional repression of Myc underlies the tumour suppressor function of AGO1 in Drosophila

    This study reports novel tumor suppressor activity for the Drosophila Argonaute family RNA-binding protein AGO1, a component of the miRNA-dependent RNA-induced silencing complex (RISC). The mechanism for growth inhibition does not, however, involve canonical roles as part of the RISC; rather, AGO1 controls cell and tissue growth by functioning as a direct transcriptional repressor of the master regulator of growth, Myc. AGO1 depletion in wing imaginal discs drives a significant increase in ribosome biogenesis, nucleolar expansion and cell growth in a manner dependent on Myc abundance. Moreover, increased Myc promoter activity and elevated Myc mRNA in AGO1-depleted animals requires RNA polymerase II transcription. Further support for transcriptional AGO1 functions is provided by physical interaction with the RNA polymerase II transcriptional machinery (chromatin remodelling factors and Mediator Complex), punctate nuclear localisation in euchromatic regions and overlap with Polycomb Group transcriptional silencing loci. Moreover, significant AGO1 enrichment is observed on the Myc promoter and AGO1 interacts with the Myc transcriptional activator Psi. Together, these data show that Drosophila AGO1 functions outside of the RISC to repress Myc transcription and inhibit developmental cell and tissue growth (Zaytseva, 2020).

    Tightly coordinated regulation of cell and tissue growth is essential for animal development. Decreased growth leads to small organs and diminished body size, whereas heightened proliferative growth is associated with genomic instability and cancer. The MYC transcription factor and growth regulator has been studied extensively since its identification as an oncogene in the early 1980s, when MYC overexpression caused by chromosomal translocation was found to drive malignant transformation in Burkitt's lymphoma. Research in subsequent decades implicated increased MYC in progression of most tumours. In normal adult tissues, MYC expression is relatively low and generally restricted to cells with regenerative and proliferative potential. Even small increases in MYC abundance are sufficient to promote proliferative cell growth; thus, understanding the molecular control of MYC expression can provide crucial insight into the mechanisms of MYC dysregulation in cancer (Zaytseva, 2020).

    In normal cells, MYC is regulated by signalling inputs from a diverse array of developmental and growth signalling pathways. The many cellular signalling inputs converging on MYC transcription are integrated by FUBP1, a KH domain protein that binds single-stranded DNA and interacts with the general transcription factor complex TFIIH to modulate MYC promoter output. The mammalian FUBP family comprises three proteins (FUBP1-3) that are represented by one orthologue in Drosophila, P-element somatic inhibitor (Psi). Like FUBP1, Psi also interacts with the RNA polymerase II (RNA Pol II) transcriptional machinery, particularly the transcriptional Mediator (MED) complex, to pattern Myc transcription and cell and tissue growth in the Drosophila wing epithelium (Guo, 2016). In addition to roles in transcription, Psi binds RNA via the KH domains and interacts with the spliceosome to regulate mRNA splicing. Although co-immunoprecipitation (co-IP) mass spectrometry detected Psi in complex with the Argonaute protein AGO1, the potential significance of this interaction is unknown (Zaytseva, 2020).

    Argonaute proteins comprise the core of the RNA-induced silencing complex (RISC), which uses noncoding RNA as a guide to target mRNAs for post-transcriptional gene silencing. Drosophila AGO2 is best characterised as part of the siRNA-induced silencing complex (siRISC), whereas AGO1 predominantly functions in microRNA-induced silencing complexes (miRISCs) and post-transcriptional mRNA silencing. Of importance to this study, AGO1-mediated mRNA silencing has been implicated in transcript destabilisation and translational repression of Myc in flies and humans. This study reports a novel role for AGO1 as a direct Myc transcriptional repressor and demonstrates that this underlies cell growth inhibition. AGO1 depletion not only increases Myc promoter activity, mRNA and protein abundance, but the elevated Myc expression requires RNA Pol II transcriptional activity. Localisation to the nucleus, together with interaction with transcriptional machinery and significant AGO1 enrichment on the Myc promoter suggests, in addition to the established roles in miRNA silencing in the cytoplasm, AGO1 constrains Myc transcription to control cell and tissue growth during Drosophila development (Zaytseva, 2020).

    This study demonstrates a novel role for AGO1 as a growth inhibitor in Drosophila. AGO1 depletion was sufficient to increase Myc (mRNA and protein) to drive ribosome biogenesis, nucleolar expansion and cell growth in a Myc and Psi-dependent manner. The increased Myc promoter activity in AGO1 knockdown wing discs, together with the α-amanitin-dependent increase in Myc pre-mRNA abundance, suggests that AGO1 represses Myc at the level of transcription. In accordance with the observed growth inhibitory capacity of AGO1, the increased Myc mRNA and protein abundance in AGO1 knockdown wings were associated with increased Myc function (i.e. activation of established Myc targets). Interestingly, although Psi co-knockdown only modestly decreased Myc mRNA levels in AGO1-depleted wings, Psi co-depletion strongly reduced expression of Myc targets. This observation suggests that Psi is not only required for Myc transcription but may also be required for activation of Myc growth targets in the context of AGO1 depletion. Thus, future studies are required to determine whether Psi and Myc bind common targets and whether Psi is required for transcriptional activation of Myc target genes (Zaytseva, 2020).

    Recent genome-wide functional RNAi screens in Drosophila S2 cells, identifying AGO1 as a modifier of Polycomb foci, suggested extra-miRNA functions for AGO1. PcG mediates epigenetic repression of key developmental genes to control cell fate, and PcG repression is stabilised via aggregation of PcG foci in the nucleus. AGO1 depletion disrupted nuclear organisation and reduced the intensity of Pc foci, suggesting that AGO1 negatively regulates PcG-mediated silencing. The Drosophila PcG complex has been characterised for roles in silencing homeotic genes by binding PcG response elements (PREs), including the Fab-7 PRE-containing regulatory element from the Hox gene, Abdominal-B. Components of the RNAi machinery, including AGO1 and Dicer-2, have been implicated in driving PcG-dependent silencing between remote copies of the Fab-7 element, engineered throughout the genome to monitor long-distance gene contacts. Interactions between Hox genes silenced by PcG proteins were decreased in AGO1 mutants, suggesting that AGO1 regulates nuclear organisation, at least in part, by stabilising PcG protein recruitment to chromatin (Zaytseva, 2020).

    Myc transcriptional autorepression, modelled in the Drosophila embryo via overexpression of Myc from an exogenous promoter, leads to repression of the endogenous Myc locus in a Pc-dependent manner . This, together with the partial overlap between AGO1 and PcG in wing imaginal disc cells, suggests that Pc mediates transcriptional autorepression of Myc via AGO1. In contrast to the current studies, where AGO1 depletion phenotypes are associated with a moderate (>three- to fivefold) increase in Myc, autoregulation in the embryo was investigated in response to non-physiological increases in Myc (over 100 times endogenous levels). Thus, the current data suggest that AGO1 binds the Myc promoter under normal conditions and is required for repression of endogenous Myc transcription, but whether AGO1 is required for Pc-dependent Myc autorepression requires further investigation. In a similar vein, super-enhancers control human MYC transcription via CTCF in the context of high-MYC cancers. Thus, failed Pc-dependent autorepression and/or defective repression of super-enhancers via CTCF could further elevate MYC to promote cancer progression. Given the observed overlap between AGO1 and Pc/CTCF in the Drosophila wing, future studies determining whether AGO1 interacts with Pc and/or CTCF to control autoregulatory feedback on Myc transcription in the context of tumorigenesis will be of great interest (Zaytseva, 2020).

    The question remains regarding how AGO1 targets Myc transcription. The physical and genetic interaction between Psi and AGO1, and the observation that AGO1 loss-of-function mutants restore cell and tissue growth in the Psi knockdown wing, suggests that AGO1 inhibits growth that is dependent on this Myc transcriptional regulator. AGO2 has been implicated in insulator-dependent looping interactions defining 3D transcriptional domains (TADs) through association with CTCF binding sites in Drosophila. Although similar roles for AGO1 have not been reported, the cancer-related super-enhancers for the MYC oncogene lie within the 2.8 Mb TAD and control MYC transcription via a common and conserved CTCF binding site located 2 kb upstream of the MYC promoter; that is, in proximity with the FUSE (1.7 kb upstream) bound by FUBP1. Moreover, gene disruption of the enhancer-docking site reduces CTCF binding and super-enhancer interaction, which results in reduced MYC expression and proliferative cell growth. AGO1 ChIP revealed significant enrichment on the Myc promoter, suggesting that AGO1 probably interacts with Psi and the RNA Pol II machinery to directly regulate Myc transcription. Given the high level of conservation between AGO and CTCF proteins throughout evolution, it is of great interest to determine whether human AGO1 also interacts with FUBP1 to regulate transcription of the MYC oncogene (Zaytseva, 2020).

    This study has shown that AGO1 behaves as a growth inhibitor during Drosophila development, through the ability to suppress Myc transcription, ribosome biogenesis and cell growth in the wing disc epithelium. Consistent with AGO1 having tumour suppressor activity, across a wide range of human cancers, large scale genomics data in cBioPortal identified AGO1 as frequently mutated or deleted in a diverse variety of tumours (e.g. reproductive, breast, intestinal, bladder, and skin cancers). Region 1p34-35 of chromosome 1, which includes AGO1, is frequently deleted in Wilms' tumours and neuro-ectodermal tumours. In neuroblastoma cell lines, AGO1 behaves as a tumour suppressor, with overexpression heightening checkpoint sensitivity and reducing cell cycle progression. GEO Profile microarray data inversely correlates AGO1 expression with proliferative index; that is, AGO1 levels are significantly lower in tumorigenic cells than in differentiated cells. In the context of cancer, it is important to determine whether AGO1 loss of function alters MYC-dependent cancer progression and vice versa. As increased abundance of the MYC oncoprotein is associated with the pathogenesis of most human tumours, deciphering such novel mechanisms of MYC repression is fundamental to understanding MYC-dependent cancer initiation and progression (Zaytseva, 2020).

    Sequential Ras/MAPK and PI3K/AKT/mTOR pathways recruitment drives basal extrusion in the prostate-like gland of Drosophila

    One of the most important but less understood step of epithelial tumourigenesis occurs when cells acquire the ability to leave their epithelial compartment. This phenomenon, described as basal epithelial cell extrusion (basal extrusion), represents the first step of tumour invasion. However, due to lack of adequate in vivo model, implication of emblematic signalling pathways such as Ras/Mitogen-Activated Protein Kinase (MAPK) and phosphoinositide 3 kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signalling pathways, is scarcely described in this phenomenon. This paper reports a unique model of basal extrusion in the Drosophila accessory gland. There, it was demonstrated that both Ras/MAPK and PI3K/AKT/mTOR pathways are necessary for basal extrusion. Furthermore, as in prostate cancer, this study shows that these pathways are co-activated. This occurs through set up of Epidermal Growth Factor Receptor (EGFR) and Insulin Receptor (InR) dependent autocrine loops, a phenomenon that, considering human data, could be relevant for prostate cancer (Rambur, 2020).

    Worldwide, a large majority of cancers originates from epithelial tissues such as lung, breast and prostate1. Despite reinforced prevention, most of the tumours are detected at late stages, and patient care is centred on invasive adenocarcinomas, resistant forms of these carcinomas and metastatic carcinomas. As late stages of cancer progression have been under intense scrutiny in the last decades, the molecular mechanisms associated to such progression are largely described, showing for example the major role of receptor tyrosine kinase (RTK)-dependent signalling pathways in these mechanisms. Typically, for prostate adenocarcinoma, the second most common cancer in men, both PI3K/AKT/mTOR pathway and Ras/MAPK pathways are associated with tumour progression. In the prostate adenocarcinoma, Ras/MAPK and PI3K/AKT/mTOR pathways display activating genetic alterations in more than 40% of primary tumours and in virtually all metastatic prostate tumours, and phosphoproteomic studies confirmed a strong correlation in the activation of these two pathways. Furthermore, pre-clinical mouse models reproducing alteration of either one or the other pathway in the prostate epithelium display tumourigenesis that mimics histopathological features of the human adenocarcinoma. Moreover, advanced tumour progression is obtained when combining alterations in both pathways. These different data emphasise that in one hand, Ras/MAPK or PI3K/AKT/mTOR pathways can initiate prostate tumour development, and in the other hand that these pathways are implicated in late phases of tumour progression. However, they explain neither their respective or combined role in actual adenocarcinoma formation nor the molecular mechanisms that could couple these two pathways to promote this phenomenon in vivo (Rambur, 2020).

    Adenocarcinoma formation occurs when pre-invasive epithelial cells acquire the ability to leave their epithelial compartment. This implicates that these cells are able to extrude from the normal epithelium and to cross the basement membrane which is the limit of the epithelial compartment. These phenomena can be described as basal extrusion and are resulting in early invasion, as opposed to late invasion associated to the metastatic process. Due to the difficulty to precisely visualise basal extrusion in animals, mechanistic associated to this phenomenon has been essentially described in cellular models or in developing tissues such as Drosophila imaginal disc of zebrafish embryo, even though the role of P120 catenin in basal extrusion has been shown in a mouse model of pancreatic neoplasia. The role of Ras/MAPK pathway in basal extrusion has only been described through the use of RasV12 as an oncogenic hit, and role of PI3K/AKT/mTOR pathway has never been assessed (Rambur, 2020).

    To determine the role of Ras/MAPK and PI3K/AKT/mTOR pathways in basal extrusion and understand the underlying mechanisms that may coordinate their hyperactivation in prostate cancer, this study has developed a new model of in vivo early invasive adenocarcinoma in the Drosophila prostate-like accessory gland. Drosophila is a powerful genetic model where more than 70% of genes implicated in human diseases display orthologs and where Ras/MAPK and PI3K/AKT/mTOR signalling pathways are well conserved. Drosophila has already proven its pertinence as cancer model for brain, lung, and colon. The Drosophila accessory gland is a functional equivalent for the prostate, playing a role in fertility by secreting seminal fluid. Secretions come from a monolayer of epithelial cells that are well differentiated and quiescent at the adult age, and there is no evidence of stem cells in this tissue. Considering that a majority of prostate adenocarcinoma is thought to originate from luminal cells, epithelial cells from the accessory gland represent a valuable model to study the mechanisms of epithelial prostate tumourigenesis (Rambur, 2020).

    This study describes this unique model of basal extrusion and tumour formation in the accessory gland that recapitulates most aspects of cancer development. Both Ras/MAPK and PI3K/Akt/TOR pathways are overactivated in the produced tumours, and these pathways cooperate to induce basal extrusion and subsequent tumour formation. Furthermore, the mechanism is described that allows the coactivation of these pathways, which relies on the sequential recruitment of a double autocrine feedback loop dependent on Epidermal (EGF/Spitz) and Insulin-like (IGF/Ilp6) Growth Factors and their respective receptors. Finally, using publicly available data of prostate cancer samples and migration assay in human pre-tumoural prostate epithelial cell line, the possible role of these findings in the actual human pathology is assessed (Rambur, 2020).

    To faithfully reproduce what is thought to happen in the earliest stages of tumour formation in patients, a single genetic alteration was produced in few clones of randomly selected and mostly differentiated cells. Furthermore, accessory gland epithelium, shown to be adjacent to a basement membrane, is surrounded by a stromal-like sheet of muscle fibres, and oncogene-induced epithelial cells are able to cross both layers to form external tumours. This recapitulates the phenomenon of basal epithelial cell extrusion, which is thought to be central to cell invasion. Basal extrusion has been described in cell culture, in Drosophila imaginal discs, in zebrafish embryos and in mouse. However, implication of Ras/MAPK and PI3K/AKT/mTOR pathways has never been assessed in this phenomenon, despite the fact that these pathways are among the most deregulated in cancers, and especially in epithelial cancers such as prostate adenocarcinoma. This study shows in a new model of accessory gland tumourigenesis that both pathways are implicated in basal extrusion, indicating that this step demands a particular state of activation for the cell that undergoes this basal extrusion. Furthermore, this finding correlates with the fact that the two considered pathways are already frequently co-deregulated in primary tumours. From these experiments, where oncogene expression is restricted to few cells and intra-tumoural inhibition of the pathways decreases invasion, it is infered that the mechanisms of basal cell extrusion are cell autonomous, as previously shown in cell lines. Indeed, this study shows that this cell-autonomous mechanism relies on the production of two growth factors, and subsequent activation of two autocrine loops. Role of autocrine loops has been hypothetized in late tumourigenesis, as higher levels of growth factors have been found in tumoural tissues, and has been studied in cell models where inhibition of these loops decreases tumourigenic features such as migration or proliferation capacity as their activation have been linked to transformation of various epithelial cells. However, a role of autocrine loops has never been demonstrated for basal extrusion in vivo. If these loops seem implicated in tumour late progression, so could they be more important for early human tumour development. In fact, many strategies have been attempted to treat cancer patients especially by blocking EGF/EGFR autocrine loop. However, for advanced prostate cancer, these strategies have shown poor results, as well for monotherapies as for combined treatments with classical anti-prostate cancer agents. It could be logical if autocrine loops are less implicated in late stages of cancer but more in the capacity for tumour cells to leave the epithelial compartment. In later stages, higher rates of activating mutations in the Ras/MAPK and PI3K/AKT/mTOR pathways could suppress the need for RTK-driven activation. In contrast, in early tumourigenesis, as fewer genetic alterations are present, activation of signalling pathways must rely on different mechanisms. As is shown in the accessory gland, this recruitment could be efficiently done in tumour cells by autocrine production of growth factors, autocrine activation of their RTK and subsequent activation of the pathways necessary for the tumour development. In a human cohort of prostate cancer samples, it was found that EGF is more expressed in primary tumours than either in normal tissue or in metastases. This could correlate with an early requirement for such growth factor in the formation of adenocarcinoma. Contrary to the observations in Drosophila, no early overexpression of IGFs can be detected in human samples. However, in human, EGFR is able to recruit both Ras/MAPK and PI3K/AKT/mTOR pathway, and EGF overexpression could drive their activation and act in the same way as EGF/Spitz and IGF/Ilp6 in Drosophila (Rambur, 2020).

    To study early phases of tumourigenesis remains difficult in vivo, especially for epithelial cells that can develop into benign tumours still in the epithelial compartment such as benign prostatic intraepithelial neoplasia, or into adenocarcinoma that are characterised by an expansion out of the epithelial compartment. The model developed in the Drosophila accessory gland represents a unique in vivo model to explore basal extrusion and early invasion. Two major pathways of cancer progression are implicated in this basal extrusion, and these two pathways are co-recruited by autocrine loops. Further investigation will be necessary to test whether other pathways implicated in late tumourigenesis are important in this phenomenon. Furthermore, it will also be important to determine which genes are activated or inhibited by these pathways and which mechanisms are recruited to promote the actual extrusion (Rambur, 2020).

    Hyperinsulinemia drives epithelial tumorigenesis by abrogating cell competition

    Metabolic diseases such as type 2 diabetes are associated with increased cancer incidence. This study shows that hyperinsulinemia promotes epithelial tumorigenesis by abrogating cell competition. In Drosophila eye imaginal epithelium, oncogenic scribble (scrib) mutant cells are eliminated by cell competition when surrounded by wild-type cells. Through a genetic screen, this study found that flies heterozygous for the insulin receptor substrate chico allow scrib cells to evade cell competition and develop into tumors. Intriguingly, chico is required in the brain's insulin-producing cells (IPCs) to execute cell competition remotely. Mechanistically, chico downregulation in IPCs causes hyperinsulinemia by upregulating a Drosophila insulin Dilp2, which activates insulin-mTOR signaling and thus boosts protein synthesis in scrib cells. A diet-induced increase in insulin levels also triggers scrib tumorigenesis, and pharmacological repression of protein synthesis prevents hyperinsulinemia-induced scrib overgrowth. These findings provide an in vivo mechanistic link between metabolic disease and cancer risk via systemic regulation of cell competition (Sanaki, 2020).

    Metabolic diseases such as type 2 diabetes and obesity are often accompanied by hyperinsulinemia, which is characterized by high levels of circulating insulin. In epidemiology, hyperinsulinemia has been implicated in increased cancer incidence. For instance, the risk of liver, pancreas, endometrium, kidney, and bladder cancers increases 1.5- to 2-fold in people with hyperinsulinemia. Although previous studies in Drosophila and rodents unveiled some aspects of the mechanism by which hyperinsulinemia promotes tumor growth and malignancy, the underlying mechanisms are still largely unknown (Sanaki, 2020).

    Most cancers originate from epithelial cells that frequently lose apicobasal polarity during tumor progression. In Drosophila imaginal epithelium, loss-of-function mutations in evolutionarily conserved apicobasal polarity genes, such as scrib or discs large (dlg), disrupt epithelial integrity and result in tumorous overgrowth. Intriguingly, such oncogenic polarity-deficient cells do not overproliferate but are eliminated from the tissue when surrounded by wild-type cells, a phenomenon called tumor-suppressive cell competition. Previous work found multiple mechanisms that drive this cell elimination via cell-cell interaction between scrib and wild-type cells, which include Sas-PTP10D ligand-receptor interaction, Slit-Robo2-Ena/VASP-mediated scrib cell extrusion, and engulfment of scrib cells by wild-type cells. Through a genetic screen in Drosophila, this study found an unexpected new regulatory mechanism whereby hyperinsulinemia systemically abrogates tumor-suppressive cell competition and thus causes tumorigenesis in the epithelium. These data could provide a mechanistic explanation for the epidemiological evidence that links hyperinsulinemia and cancer incidence, thus contributing to a better understanding of cancer biology in vivo (Sanaki, 2020).

    This study found that hyperinsulinemia in flies systemically suppresses cell competition in the eye epithelium, leading to tumorous overproliferation of polarity-deficient cells that are normally eliminated when surrounded by wild-type cells. It has been reported that high-sugar diet promotes tumor growth and metastasis of fly tumors with elevated Ras and Src signaling, providing a model of how abnormal physiology promotes tumor progression. In addition, studies in mice have shown that high-fat diet-induced obesity suppresses extrusion of oncogenic RasV12-expressing cells from mice intestine and that endogenous hyperinsulinemia contributes to pancreatic ductal adenocarcinoma. Thus, abnormal physiology, especially hyperinsulinemia, has a promotive effect on tumor development and progression, yet the mechanism by which hyperinsulinemia controls the initial step of tumorigenesis has been unclear. The current observations indicate that chico heterozygous mutant or IPCs-specific chico-knockdown larvae can be used as a Drosophila model of hyperinsulinemia. Consistently, although chico homozygous mutant flies drastically decrease their body weight, chico heterozygous mutant flies show increased body weight, implying a phenotypic outcome of hyperinsulinemia (Sanaki, 2020).

    The findings that hyperinsulinemia systemically abrogates tumor-suppressive cell competition by boosting InR-TOR-mediated protein synthesis in pre-malignant cells may provide an in vivo mechanistic link between metabolic diseases and cancer risk. Previous work has shown that Sas-PTP10D signaling in scrib cells promotes their elimination by repressing epidermal growth factor receptor (EGFR) signaling. Defects in Sas-PTP10D signaling attenuates scrib cell elimination via cooperation between EGFR-Ras and TNF-JNK signaling, which leads to activation of the Hippo pathway effector Yorkie (Yki). On the other hand, this study found that hyperinsulinemia attenuates scrib cell elimination by fueling insulin-mTor signaling. Given that these two signaling pathways are independent, there would be no direct cross talk between Sas-PTP10D signaling and hyperinsulinemia-driven tumorigenesis. Rather, it is possible that both Sas-PTP10D inactivation (Yki activation) and insulin signaling activation (Tor activation) lead to the same biological outcome, namely, elevation of protein synthesis, which could explain how insulin signaling overrides Sas-PTP10D signaling (Sanaki, 2020).

    Notably, differential levels of protein synthesis between cells has long been implicated in regulating classical Minute cell competition, which is a competitive elimination of cells with a heterozygous mutation for a ribosomal protein gene. In addition, recent work has found that losers of cell competition triggered by different mutations such as Minute, Myc, Mahjong, and Hel25E commonly show lower protein synthesis levels than that neighboring winners do (Nagata, 2019). Moreover, insulin-TOR signaling has been shown to control cell competition during mouse embryonic development. These observations suggest that differential levels of insulin-TOR signaling and protein synthesis between cells are the key for cell competition. Supporting this notion, scrib-induced cell competition can be compromised either by introducing Minute mutation in wild-type winners or by overexpressing Myc in scrib losers. These data show that scrib cells are insensitive to environmental insulin and thus are lower in insulin-TOR signaling and protein synthesis levels compared with that of the neighbors, and hyperinsulinemia reverses this balance and causes scrib tumorigenesis. Given that a drug treatment targeting cellular metabolism could prevent hyperinsulinemia-driven tumorigenesis, cancer risk risen by metabolic diseases may become controllable in the future (Sanaki, 2020).

    Snail-induced claudin-11 prompts collective migration for tumour progression

    Epithelial-mesenchymal transition (EMT) is a pivotal mechanism for cancer dissemination. However, EMT-regulated individual cancer cell invasion is difficult to detect in clinical samples. Emerging evidence implies that EMT is correlated to collective cell migration and invasion with unknown mechanisms. This study shows that the EMT transcription factor Snail elicits collective migration in squamous cell carcinoma by inducing the expression of a tight junctional protein, claudin-11. Mechanistically, tyrosine-phosphorylated claudin-11 activates Src, which suppresses RhoA activity at intercellular junctions through p190RhoGAP (see Drosophila RhoGAPp190), maintaining stable cell-cell contacts. In head and neck cancer patients, the Snail-claudin-11 (see Drosophila Snail) axis prompts the formation of circulating tumour cell clusters, which correlate with tumour progression. Overexpression of snail correlates with increased claudin-11, and both are associated with a worse outcome. This finding extends the current understanding of EMT-mediated cellular migration via a non-individual type of movement to prompt cancer progression (Li, 2019).

    The expression and functional impact of different claudins are distinct among different cancers. Snail did not influence the expression of other claudins in the 2.5D system of squamous cell carcinoma (SCC). Snail-regulated claudin-11 did not interfere with either cell viability or single-cell migration, but it did modulate collective migration and invasion, lymph node metastases and clustering circulating tumor cell (CTC) formation. It is suggested that during tumour metastasis, claudin-11 contributes to the maintenance of cell-cell contacts to enhance metastatic efficiency. Intriguingly, claudin-11 not only acted as an adhesive protein, but importantly, recruited Src-phosphorylated p190RhoGAP to inactivate RhoA at intercellular junctions. It is also noted that the triggering event for claudin-11-mediated collective migration is the phosphorylation of tyrosine residues in the C terminus of claudin-11. Although it is noted that suppression of FAK reduces claudin-11 phosphorylation, whether FAK is indeed indispensable for claudin-11 Tyr 191/Tyr 192 phosphorylation requires further confirmation (Li, 2019).

    Recent studies suggest that a fraction of CTCs travel as clusters and they exhibit a greater metastatic potential than single CTCs. This study showed that the Snail-claudin-11 axis correlates with the number of CTC clusters in a prospective HNSCC cohort. However, a concern for the currently available methods of CTC enumeration is that most of them are antibody-dependent and nonspecific binding cannot be totally excluded. In the current series, although the applied microfluidic platform has been reported to carry a higher sensitivity in capturing CTCs, it should be noted that the enumeration results could be regarded only as an association study before genomic validation of all captured CTCs. However, validating genomic alterations in captured CTCs of all cases will not be generally affordable for routine clinical practices. Another issue is that the correlation between the expression of Snail/claudin-11 in primary tumours and the number of clustering CTCs was weak in these cases. A possible explanation for this discrepancy is that there was a lag between the sampling dates of the primary tumour and the CTCs in these patients, which implies that the pathobiology of the primary tumours, collected at a different time from the CTCs, may not reflect the characteristics of the CTCs (Li, 2019).

    In summary, this study demonstrated the mechanism of collective migration and generation of CTC clusters in SCC, which not only extends understanding of the mechanisms and routes of EMT-mediated cancer dissemination but also provides potential targets for preventing the spread of SCC (Li, 2019).

    Cross-species identification of PIP5K1-, splicing- and ubiquitin-related pathways as potential targets for RB1-deficient cells

    The RB1 tumor suppressor is recurrently mutated in a variety of cancers including retinoblastomas, small cell lung cancers, triple-negative breast cancers, prostate cancers, and osteosarcomas. Finding new synthetic lethal (SL) interactions with RB1 could lead to new approaches to treating cancers with inactivated RB1. This study identified 95 SL partners of RB1 based on a Drosophila screen for genetic modifiers of the eye phenotype caused by defects in the RB1 ortholog, Rbf1. 38 mammalian orthologs of Rbf1 modifiers were evaluated as RB1 SL partners in human cancer cell lines with defective RB1 alleles. It was further shown that for many of the RB1 SL genes validated in human cancer cell lines, low activity of the SL gene in human tumors, when concurrent with low levels of RB1 was associated with improved patient survival. Higher order combinatorial gene interactions were investigated by creating a novel Drosophila cancer model with co-occurring Rbf1, Pten and Ras mutations; targeting RB1 SL genes in this background suppressed the dramatic tumor growth and rescued fly survival whilst having minimal effects on wild-type cells. Finally, it was found that drugs targeting the identified RB1 interacting genes/pathways, such as UNC3230, PYR-41, TAK-243, isoginkgetin, madrasin, and celastrol also elicit SL in human cancer cell lines. In summary, this study identified several high confidence, evolutionarily conserved, novel targets for RB1-deficient cells that may be further adapted for the treatment of human cancer (Parkhitko, 2021).

    Cooperation between oncogenic Ras and wild-type p53 stimulates STAT non-cell autonomously to promote tumor radioresistance

    Oncogenic RAS mutations are associated with tumor resistance to radiation therapy. Cell-cell interactions in the tumor microenvironment (TME) profoundly influence therapy outcomes. However, the nature of these interactions and their role in Ras tumor radioresistance remain unclear. This study used Drosophila oncogenic Ras tissues and human Ras cancer cell radiation models to address these questions. It was discovered that cellular response to genotoxic stress cooperates with oncogenic Ras to activate JAK/STAT non-cell autonomously in the TME. Specifically, p53 is heterogeneously activated in Ras tumor tissues in response to irradiation. This mosaicism allows high p53-expressing Ras clones to stimulate JAK/STAT cytokines, which activate JAK/STAT in the nearby low p53-expressing surviving Ras clones, leading to robust tumor re-establishment. Blocking any part of this cell-cell communication loop re-sensitizes Ras tumor cells to irradiation. These findings suggest that coupling STAT inhibitors to radiotherapy might improve clinical outcomes for Ras cancer patients (Dong, 2021).

    Misshapen Disruption Cooperates with Ras(V12) to Drive Tumorigenesis

    Although RAS family genes play essential roles in tumorigenesis, effective treatments targeting RAS-related tumors are lacking, partly because of an incomplete understanding of the complex signaling crosstalk within RAS-related tumors. A large-scale genetic screen in Drosophila eye imaginal discs identified Misshapen (Msn) as a tumor suppressor that synergizes with oncogenic Ras (Ras(V12)) to induce c-Jun N-terminal kinase (JNK) activation and Hippo inactivation, then subsequently leads to tumor overgrowth and invasion. Moreover, ectopic Msn expression activates Hippo signaling pathway and suppresses Hippo signaling disruption-induced overgrowth. Importantly, it was further found that Msn acts downstream of protocadherin Fat (Ft) to regulate Hippo signaling. Finally, msn as a Yki/Sd target gene that regulates Hippo pathway in a negative feedback manner. Together, these findings identified Msn as a tumor suppressor and provide a novel insight into RAS-related tumorigenesis that may be relevant to human cancer biology (Kong, 2021).

    The level of oncogenic Ras determines the malignant transformation of Lkb1 mutant tissue in vivo

    The genetic and metabolic heterogeneity of RAS-driven cancers has confounded therapeutic strategies in the clinic. To address this, rapid and genetically tractable animal models are needed that recapitulate the heterogeneity of RAS-driven cancers in vivo. This study generate a Drosophila melanogaster model of Ras/Lkb1 mutant carcinoma. Low-level expression of oncogenic Ras (RasLow) was shown to promote the survival of Lkb1 mutant tissue, but results in autonomous cell cycle arrest and non-autonomous overgrowth of wild-type tissue. In contrast, high-level expression of oncogenic Ras (RasHigh) transforms Lkb1 mutant tissue resulting in lethal malignant tumors. Using simultaneous multiview light-sheet microcopy, this study has characterized invasion phenotypes of Ras/Lkb1 tumors in living larvae. This molecular analysis reveals sustained activation of the AMPK pathway in malignant Ras/Lkb1 tumors, and demonstrate the genetic and pharmacologic dependence of these tumors on CaMK-activated Ampk. LKB1 mutant human lung adenocarcinoma patients with high levels of oncogenic KRAS were shown to exhibit worse overall survival and increased AMPK activation. These results suggest that high levels of oncogenic KRAS is a driving event in the malignant transformation of LKB1 mutant tissue, and uncovers a vulnerability that may be used to target this aggressive genetic subset of RAS-driven tumors (Rackley, 2021).

    Aneuploidy facilitates dysplastic and tumorigenic phenotypes in the Drosophila gut

    Aneuploidy has been strongly linked to cancer development, and published evidence has suggested that aneuploidy can have an oncogenic or a tumor suppressor role depending on the tissue context. Using the Drosophila midgut as a model, it was recently described that adult intestinal stem cells (ISCs), do not activate programmed cell death upon aneuploidy induction, leading to an increase in ISC proliferation rate, and tissue dysplasia.  How aneuploidy impacts ISCs in intestinal tumorigenic models remains to be investigated, and it represents a very important biological question to address since data from multiple in vivo models suggests that the cellular impact of aneuploidy is highly dependent on the cellular and tissue context. Using manipulation of different genetic pathways such as EGFR, JAK-STAT and Notch that cause dysplastic phenotypes in the Drosophila gut, this study found that concomitant aneuploidy induction by impairment of the Spindle Assembly Checkpoint (SAC) consistently leads to a more severe progression of intestinal dysplasia or tumorigenesis. This is characterized by an accumulation of progenitor cells, high tissue cell density and higher stem cell proliferation rates, revealing an additive or synergistic effect depending on the misregulated pathway in which aneuploidy was induced. Thus, these data suggests that in the Drosophila gut, both dysplasia and tumorigenic phenotypes can be fueled by inducing genomic instability of resident stem cells (Bras, 2021).

    Methionine restriction breaks obligatory coupling of cell proliferation and death by an oncogene Src in Drosophila

    Oncogenes often promote cell death as well as proliferation. How oncogenes drive these diametrically opposed phenomena remains to be solved. A key question is whether cell death occurs as a response to aberrant proliferation signals or through a proliferation-independent mechanism. This study revealed that Src, the first identified oncogene, simultaneously drives cell proliferation and death in an obligatorily coupled manner through parallel MAPK pathways. The two MAPK pathways diverge from a lynchpin protein Slpr. A MAPK p38 drives proliferation whereas another MAPK JNK drives apoptosis independently of proliferation signals. Src-p38-induced proliferation is regulated by methionine-mediated Tor signaling. Reduction of dietary methionine uncouples the obligatory coupling of cell proliferation and death, suppressing tumorigenesis and tumor-induced lethality. These findings provide an insight into how cells evolved to have a fail-safe mechanism that thwarts tumorigenesis by the oncogene Src. This study also exemplifies a diet-based approach to circumvent oncogenesis by exploiting the fail-safe mechanism (Nishida, 2021).

    The H3.3K27M oncohistone antagonizes reprogramming in Drosophila

    Development proceeds by the activation of genes by transcription factors and the inactivation of others by chromatin-mediated gene silencing. In certain cases development can be reversed or redirected by mis-expression of master regulator transcription factors. This must involve the activation of previously silenced genes, and such developmental aberrations are thought to underlie a variety of cancers. This study expressed the wing-specific Vestigial master regulator to reprogram the developing eye, and test the role of silencing in reprogramming using an H3.3K27M oncohistone mutation that dominantly inhibits histone H3K27 trimethylation. Production of the oncohistone was found to block eye-to-wing reprogramming. CUT&Tag chromatin profiling of mutant tissues shows that H3K27me3 of domains is generally reduced upon oncohistone production, suggesting that a previous developmental program must be silenced for effective transformation. Strikingly, Vg and H3.3K27M synergize to stimulate overgrowth of eye tissue, a phenotype that resembles that of mutations in Polycomb silencing components. Transcriptome profiling of elongating RNA Polymerase II implicates the mis-regulation of signaling factors in overgrowth. These results demonstrate that growth dysregulation can result from the simple combination of crippled silencing and transcription factor mis-expression, an effect that may explain the origins of oncohistone-bearing cancers (Ahmad, 2021).


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    Zygotically transcribed genes

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